Gram-positive bacteria specific binding compounds

ABSTRACT

The present invention provides improved binding compounds capable of specifically binding Gram-positive bacteria. Binding compounds are provided that are fully human, enabling therapeutic applications in human individuals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/837,358, filed Jul. 15, 2010 and claims priority to European PatentApplication No. 09165558.9, filed Jul. 15, 2009, and U.S. ProvisionalPatent Application No. 61/225,878, filed Jul. 15, 2009, the disclosuresof which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The invention relates to the fields of biology, immunology and medicine.

BACKGROUND OF THE INVENTION

Gram-positive microorganisms cause the majority of systemic infections.One important member of these Gram-positive pathogens is Staphylococcusaureus (S. aureus). About 20% of the population is a long-term carrierof S. aureus. S. aureus can cause a range of illnesses from minor skininfections, such as pimples, impetigo (may also be caused byStreptococcus pyogenes), boils, cellulitis folliculitis, furuncles,carbuncles, scalded skin syndrome and abscesses, to life-threateningdiseases such as pneumonia, meningitis, osteomyelitis, endocarditis,Toxic shock syndrome (TSS), and septicemia. S. aureus is capable ofinfecting all kinds of organs and tissues. S. aureus infections occur inimmunocompetent as well as immune compromised people. About 50% of theinfections in US intensive care units are caused by this pathogen. Threehundred thousand S. aureus infections per year, resulting in 12,000deaths, are reported in the US (see also Moran et al. NEMJ 355, 666-674(2006)).

A major problem is the increasing antibiotic resistance of S. aureus.Methicillin-resistant S. aureus (MRSA) emerged in the 1960s. It wasinitially identified in health care settings. However, MRSA appears tobe present among persons in the community which have not beenhospitalized. Treatment of MRSA is difficult and expensive, due to thelimited sensitivity of MRSA to antibiotics. However, very fewalternatives of antibiotics are available. Vancomycin is often used totreat penicillin-resistant MRSA. U.S. Pat. No. 6,939,543 describes amurine antibody against lipoteichoic acid (LTA) which is capable ofbinding S. aureus. Based on this murine antibody, a recombinant chimericmouse/human antibody was produced which contains human heavy and lightchain constant domains. However, such chimeric mouse/human antibody hasthe disadvantage that murine sequences are present, which involves therisk of serious side effects when administered to humans.

SUMMARY OF THE INVENTION

Provided herein are antibodies or functional parts thereof orimmunoglobulin chains or functional equivalents thereof, which comprise:(a) a heavy chain CDR1 sequence comprising a sequence which has at least70% sequence identity to the sequence RFAMS (SEQ ID NO:1), and/or (b) aheavy chain CDR2 sequence comprising a sequence which has at least 70%sequence identity to the sequence SINNGNNPYYARSVQY (SEQ ID NO:2), and/or(c) a heavy chain CDR3 sequence comprising a sequence which has at least70% sequence identity to the sequence DHPSSGWPTFDS (SEQ ID NO:3), and/or(d) a light chain CDR1 sequence comprising a sequence which has at least70% sequence identity to the sequence RASENVGDWLA (SEQ ID NO:4), and/or,(e) a light chain CDR2 sequence comprising a sequence which has at least70% sequence identity to the sequence KTSILES (SEQ ID NO:5), and/or (f)a light chain CDR3 sequence comprising a sequence which has at least 70%sequence identity to the sequence QHYXRFPYT, wherein X is I or M (SEQ IDNO:6). In some embodiments, the antibody or functional part thereof orimmunoglobulin chain or functional equivalent thereof, which comprises:(a) a heavy chain CDR1 sequence comprising a sequence which has thesequence RFAMS (SEQ ID NO:1), and/or (b) a heavy chain CDR2 sequencecomprising a sequence which has the sequence SINNGNNPYYARSVQY (SEQ IDNO:2), and/or (c) a heavy chain CDR3 sequence comprising a sequencewhich has the sequence DHPSSGWPTFDS (SEQ ID NO:3), and/or (d) a lightchain CDR1 sequence comprising a sequence which has the sequenceRASENVGDWLA (SEQ ID NO:4), and/or, (e) a light chain CDR2 sequencecomprising a sequence which has the sequence KTSILES (SEQ ID NO:5),and/or (f) a light chain CDR3 sequence comprising a sequence which hasthe sequence QHYXRFPYT, wherein X is I or M (SEQ ID NO:6).

In some embodiments, the antibody or functional part or immunoglobulinchain or functional equivalent has a heavy chain sequence comprising asequence which has at least 70% sequence identity to the sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID NO:7)and/or having a light chain sequence which has at least 70% sequenceidentity to the sequenceDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRTV, wherein X is I of M (SEQID NO:8).

In some embodiments, the antibody or functional part or immunoglobulinchain or functional equivalent has a heavy chain sequence comprising asequence which has at least 70% sequence identity to the sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID NO:7)and/or having a light chain sequence which has at least 70% sequenceidentity to the sequenceDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRA, wherein X is I of M (SEQID NO:10).

In some embodiments, the antibody or functional part or immunoglobulinchain or functional equivalent has a heavy chain sequence comprising asequence which has at least 70% sequence identity to the sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID NO:7)and/or having a light chain sequence which has at least 70% sequenceidentity to the sequenceDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKVEIKRTV, wherein X is I of M (SEQID NO:11).

In some embodiments, the antibody or functional part or immunoglobulinchain or functional equivalent has a heavy chain sequence comprising asequence which has at least 70% sequence identity to the sequenceEVQLVESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID NO:9)and/or having a light chain sequence which has at least 70% sequenceidentity to the sequenceDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRTV, wherein X is I of M (SEQID NO:8).

In some embodiments, the antibody or functional part or immunoglobulinchain or functional equivalent has a heavy chain sequence comprising asequence which has at least 70% sequence identity to the sequenceEVQLVESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID NO:9)and/or having a light chain sequence which has at least 70% sequenceidentity to the sequenceDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRA, wherein X is I of M (SEQID NO:10).

In some embodiments, the antibody or functional part or immunoglobulinchain or functional equivalent has a heavy chain sequence comprising asequence which has at least 70% sequence identity to the sequenceEVQLVESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID NO:9)and/or having a light chain sequence which has at least 70% sequenceidentity to the sequenceDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKVEIKRTV, wherein X is I of M (SEQID NO:11).

In some embodiments of any of the antibodies or functional parts orimmunoglobulin chains or functional equivalents, an asparagine has beenreplaced by another amino acid. In some embodiments, the asparagine isan asparagine at position 53 of the heavy chain, whereby the amino acidnumbering is according to Kabat (1991). In some embodiments, the otheramino acid is serine.

In some embodiments of any of the antibodies or functional parts orimmunoglobulin chains or functional equivalents, at least one amino acidother than cysteine has been replaced with cysteine. In someembodiments, the at least one amino acid other than cysteine is valineat light chain position 205 and/or valine at light chain position 110,and/or alanine at heavy chain position 114, whereby the amino acidnumbering is according to Kabat (1991).

The invention provides isolated antibodies that bind to in vivo-grown S.aureus. In some embodiments, the antibody is human.

The invention also provides antibodies or functional parts orimmunoglobulin chains or functional equivalents capable of binding anSD-repeat dependent epitope.

Also provided herein are antibodies or functional parts orimmunoglobulin chains or functional equivalents capable of binding to S.aureus ClfA, ClfB, SdrC, SdrD and SdrE.

Provided herein are antibodies or functional parts or immunoglobulinchains or functional equivalents which are capable of competing with anyantibody or functional part or immunoglobulin chain or functionalequivalent described herein for binding to a Staphylococcus species.

In some embodiments of any of the antibodies or functional parts orimmunoglobulin chains or functional equivalents, the antibody orfunctional part or immunoglobulin chain or functional equivalent is ahuman antibody.

Provided herein are further isolated, synthetic or recombinant nucleicacid sequences with a length of at least 15 nucleotides, or functionalequivalents thereof, encoding at least one CDR sequence of any of theantibodies or functional parts or immunoglobulin chains or functionalequivalents described herein.

The invention provides isolated, synthetic or recombinant nucleic acidsequences, or functional equivalents thereof, comprising a sequencewhich has at least 70% sequence identity to a sequence selected from thegroup consisting of cgctttgccatgagc (SEQ ID NO:12),tcgatcaataatgggaataacccatactacgcacggtcggtacaatac (SEQ ID NO:13),gatcaccctagtagtggctggcccacctttgactcc (SEQ ID NO:14),cgggccagtgaaaacgttggtgactggttggcc (SEQ ID NO:15), aagacatctattctagaaagt(SEQ ID NO:16) and caacactatatacgtttcccgtacact (SEQ ID NO:17). In someembodiment, the nucleic acid sequence or functional equivalent comprisesa sequence which has at least 70% sequence identity to at least part ofa nucleotide sequence as depicted in FIG. 1, said part having at least15 nucleotides and encoding at least one CDR region.

Also provided herein are isolated, synthetic or recombinant nucleic acidsequences, or functional equivalents thereof, comprising a sequenceencoding an amino acid sequence which has at least 70% sequence identityto the sequence RFAMS (SEQ ID NO:1), and/or at least 70% sequenceidentity to the sequence SINNGNNPYYARSVQY (SEQ ID NO:2), and/or at least70% sequence identity to the sequence DHPSSGWPTFDS (SEQ ID NO:3), and/orat least 70% sequence identity to the sequence RASENVGDWLA (SEQ IDNO:4), and/or at least 70% sequence identity to the sequence KTSILES(SEQ ID NO:5), and/or at least 70% sequence identity to the sequenceQHYXRFPYT, wherein X is I or M (SEQ ID NO:6), and/or at least 70%sequence identity to the sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID NO:7),and/or at least 70% sequence identity to the sequenceDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRTV, wherein X is I or M (SEQID NO:8).

Provided herein are any antibodies or functional parts or animmunoglobulin chains or functional equivalents thereof or any nucleicacid sequence or functional equivalents thereof described herein for useas a medicament and/or prophylactic agent.

Provided herein are also any antibodies or functional parts or animmunoglobulin chains or functional equivalents thereof or any nucleicacid sequences or functional equivalents thereof described herein foruse as a medicament and/or prophylactic agent for at least in parttreating and/or preventing a Gram-positive bacterium-related disorder.Methods of treating and/or preventing a Gram-positive bacterium-relateddisorder comprising administering an effective amount of any antibodiesor functional parts or an immunoglobulin chains or functionalequivalents thereof or any nucleic acid sequences or functionalequivalents thereof described herein are further provided herein.Provided are uses of any antibodies or functional parts or animmunoglobulin chains or functional equivalents thereof or any nucleicacid sequences or functional equivalents thereof described herein forthe preparation of a medicament and/or prophylactic agent for at leastin part treating and/or preventing a Gram-positive bacterium-relateddisorder.

Provided herein are pharmaceutical compositions comprising anyantibodies or functional parts or an immunoglobulin chains or functionalequivalents thereof or any nucleic acid sequences or functionalequivalents thereof described herein and a pharmaceutical acceptablecarrier, diluent or excipient.

Also provided herein are isolated or recombinant antibodies producingcell capable of producing any antibodies or functional parts orimmunoglobulin chains or functional equivalents described herein.Provided are methods for producing any antibodies or functional parts orimmunoglobulin chains or functional equivalents described herein,comprising providing a cell with any nucleic acid sequences orfunctional equivalents described herein and allowing said cell totranslate the nucleic acid sequences or functional equivalents describedherein, thereby producing any of the antibodies or functional parts orimmunoglobulin chains or functional equivalents described herein. Insome embodiments, the method further comprises harvesting, purifyingand/or isolating any of the antibodies or functional parts orimmunoglobulin chains or functional equivalents described herein.

Provided herein are uses of any antibodies or functional parts or animmunoglobulin chains or functional equivalents thereof or any nucleicacid sequences or functional equivalents thereof described herein fordiagnosis of a Staphylococcus infection. Methods for diagnosing aStaphylococcus infection comprising contacting a sample with anyantibodies or functional parts or an immunoglobulin chains or functionalequivalents thereof or any nucleic acid sequences or functionalequivalents thereof described herein are also provided. Provided arealso antibodies or functional parts thereof or an immunoglobulin chainsor functional equivalents thereof described herein for use in thediagnosis of a Staphylococcus infection.

The invention also provides use of any antibodies or functional parts oran immunoglobulin chains or functional equivalents thereof or anynucleic acid sequences or functional equivalents thereof describedherein for detecting S. aureus and/or S. epidermidis. Methods fordetecting S. aureus and/or S. epidermidis comprising contacting a samplewith any antibodies or functional parts or an immunoglobulin chains orfunctional equivalents thereof or any nucleic acid sequences orfunctional equivalents thereof described herein are also provided.

Provided are also methods for isolating S. aureus and/or S. epidermidisbacteria comprising contacting a sample (e.g. solution) with anyantibodies or functional parts or an immunoglobulin chains or functionalequivalents thereof described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Heavy chain and light chain sequences of antibody F1.

FIG. 2: F1 antibody binds to LTA preparations from several Gram-positivebacteria. (a) LTA preparations were derived from Sigma and tested in acapture ELISA with a mouse polyclonal anti-LTA or (b) a highly purifiedset of LTA preparations derived from W. Fischer. D25, a human anti-RSVantibody was used as non-specific negative control and a mousemonoclonal anti-LTA antibody as a positive control.

FIG. 3: Recombinant F1 antibody binds to S. aureus but not to S.pneumoniae. (a) Bacteria were incubated with F1 antibody, or a controlIgG (D25, a human anti-RSV antibody) or without first antibody (IgG-PEonly). After washing the secondary antibody was added (IgG-PE). (b) 6clinical isolates were tested in two separate experiments for thebinding of F1. Tested were a PVL+ strain (SA-1), 3 regular (SA-2 SA-3and SA-4), and 2 MRSA strains (SA-5 and SA-6).

FIG. 4: (a) Binding of rF1 antibody to 14 S. aureus strains. (b) rF1antibody binds to S. epidermidis but not to Bacillus subtilis,Enterococcus faecalis, Listeria monocytogenes and Streptococcuspyogenes.

FIG. 5: (a) rF1 antibody binds to MRSA in different growth stages, IsoC: isotype control, Med: media control (b) rF1 antibody binds to MRSAisolated from infected tissue.

FIG. 6: rF1 antibody binds to SDR proteins. (a) Immunoprecipitation (IP)of a commercial teichoic acid preparation of S. aureus (Wood 46 strain)with rF1 or isotype control antibody, followed by Western blotting withrF1 antibody (left) and mass spectrometry analysis of fragments from theWTA preparation bound by rF1 antibody (right), (b) Immunoprecipitation(IP) of cell wall lysate of S. aureus (USA300 strain) with rF1 orcontrol antibody, followed by Western blotting (WB) with rF1 antibody(left) and mass spectrometry analysis of cell wall fragments (USA300strain) bound by rF1 antibody (right) (c) Immunoprecipitation of cellwall lysate of S. epidermidis with rF1 or isotype control antibody,followed by Western blotting with rF1 antibody (left), mass spectrometryanalysis of cell wall fragments bound by rF1 antibody (right).

FIG. 7: Binding of rF1 to SDR proteins expressed in S. aureus and E.coli. (a) Western blotting of S. aureus and E. coli lysates containingoverexpressed His-tagged ClfA using anti-His (left) and rF1 (right)antibody. (b) Western Bloting of E. coli cell lysates containinghis-tagged ClfA, ClfB, SdrC, SdrD and SdrE following incubation with S.aureus lysate with rF1 (top) or anti-His (bottom) antibody.

FIG. 8: rF1 binds to SDR domains expressed by S. aureus. Constructsexpressed by S. aureus and tested for binding to rF1 (left), Westernblots of S. aureus lysates containing the expressed constructs withanti-MBP (maltose binding protein), anti-His and rF1 antibody (right).

FIG. 9: Sequences of heavy chain A114C (a) and light chain V205C (b)variants of antibody rF1. Numbering according to Kabat (1991), boxes:CDRs.

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide means and methodsfor counteracting and/or preventing Gram-positive bacteria-relateddiseases. It is a further object of the invention to provide alternativeand/or improved binding compounds against various Gram-positivebacteria, preferably Staphylococcus species, more preferably MRSA. It isa further object of the invention to provide human binding compoundsagainst various Gram-positive bacteria, preferably Staphylococcusspecies, more preferably MRSA.

The present invention provides antibodies, functional parts thereof orimmunoglobulin chains or functional equivalents thereof capable ofspecifically binding Staphylococcus species in their natural context.Therefore, they are useful for counteracting and/or preventing and/ordiagnosing disorders related to the presence of Staphylococcus species.Preferably, S. aureus is counteracted. In a particularly preferredembodiment, MRSA is counteracted. Another preferred Staphylococcusspecies which is counteracted is Staphylococcus epidermidis.

Staphylococcus epidermidis is usually non-pathogenic, but patients witha compromised immune system are often at risk for developing aninfection. Infections can be both hospital and community acquired, butthey are more of a threat to hospital patients. Most susceptible toinfection with S. epidermidis are intravenous drug users, newborns,elderly, and those using catheters or other artificial appliances.Infections are associated with intravascular devices (prosthetic heartvalves, shunts, etc.) but also commonly occur in prosthetic joints,catheters, and large wounds. Symptoms include fever, headache, fatigue,anorexia and dyspnea.

In a preferred embodiment, the present invention provides isolated orrecombinant human antibodies, functional parts thereof or immunoglobulinchains or functional equivalents thereof capable of specifically bindingStaphylococcus species. Human antibodies and functional parts accordingto the invention are capable of binding Staphylococcus species in itsnatural context, so that various Staphylococcus species are counteractedupon administration of said antibodies, functional parts thereof orimmunoglobulin chains or functional equivalents thereof. Said humanantibodies, functional parts thereof or immunoglobulin chains orfunctional equivalents thereof are therefore particularly suitable fortreatment or prevention of infection by such Staphylococcus species.Said human antibodies, functional parts thereof or immunoglobulin chainsor functional equivalents thereof according to the invention are moresuitable for therapeutic and/or prophylactic use for human individuals,as compared to chimeric antibodies, because of the absence of non-humansequences. This significantly reduces the risk of adverse side effects.

One particularly preferred antibody according to the present inventionis the antibody designated “F1”, which has heavy chain and light chainvariable domain sequences as depicted in FIG. 1. The term “F1” as usedherein encompasses all F1 antibodies, for instance isolated orrecombinantly produced F1. Recombinantly produced F1 is herein alsocalled “rF1”. The CDR sequences of F1, which in particular contribute tothe antigen-binding properties of F1, are also depicted in FIG. 1.Antibody F1 is fully human, is capable of specifically bindingStaphylococcus species such as S. aureus and S. epidermidis and istherefore preferred for therapeutic use for human individuals.

Importantly, antibody F1 is capable of binding whole bacteria in vivo aswell as in vitro. Further provided is therefore an isolated orrecombinant human antibody or a functional part thereof, which iscapable of specifically binding S. aureus and/or S. epidermidis.Furthermore, antibody F1 is capable of binding to bacteria that havebeen grown in infected tissue of, for example, an animal. Provided istherefore an isolated antibody that binds to in vivo-grown S. aureus,wherein “in vivo-grown” is defined as grown in infected tissue of ananimal during infection with S. aureus. Also provided is an isolated,recombinant or synthetic immunoglobulin chain or functional equivalentthereof comprising at least one CDR sequence of a human immunoglobulinvariable region which is specific for S. aureus and/or S. epidermidis.

A functional part of an antibody is defined as a part which has at leastone shared property as said antibody in kind, not necessarily in amount.Said functional part is capable of binding the same antigen as saidantibody, albeit not necessarily to the same extent. A functional partof an antibody preferably comprises a single domain antibody, a singlechain antibody, a nanobody, an unibody, a single chain variable fragment(scFv), a Fab fragment or a F(ab′)₂ fragment.

A functional part of an antibody is also produced by altering anantibody such that at least one property—preferably an antigen-bindingproperty—of the resulting compound is essentially the same in kind, notnecessarily in amount. This is done in many ways, for instance throughconservative amino acid substitution, whereby an amino acid residue issubstituted by another residue with generally similar properties (size,hydrophobicity, etc), such that the overall functioning is likely not tobe seriously affected.

As is well known by the skilled person, a heavy chain of an antibody isthe larger of the two types of chains making up an immunoglobulinmolecule. A heavy chain comprises constant domains and a variabledomain, which variable domain is involved in antigen binding. A lightchain of an antibody is the smaller of the two types of chains making upan immunoglobulin molecule. A light chain comprises a constant domainand a variable domain. The variable domain is, together with thevariable domain of the heavy chain, involved in antigen binding.

Complementary-determining regions (CDRs) are the hypervariable regionspresent in heavy chain variable domains and light chain variabledomains. The CDRs of a heavy chain and the connected light chain of anantibody together form the antigen-binding site.

A functional equivalent of an immunoglobulin chain is defined herein asan artificial binding compound, comprising at least one CDR sequence ofan immunoglobulin chain.

Now that the present invention provides the insight that the CDRsequences depicted in FIG. 1 provide desired binding characteristics, askilled person is well capable of generating variants comprising atleast one altered CDR sequence. For instance, conservative amino acidsubstitution is applied. It is also possible to alter at least one CDRsequence depicted in FIG. 1 in order to generate a variant antibody, ora functional part thereof, with at least one altered property ascompared to F1. Preferably, an antibody or functional part is providedcomprising a CDR sequence which is at least 70% identical to a CDRsequence as depicted in FIG. 1, so that the favorable bindingcharacteristics of F1 are at least in part maintained or even improved.A CDR sequence as depicted in FIG. 1 is preferably altered such that theresulting antibody or functional part comprises at least one improvedproperty, such as for instance an improved binding affinity, selectivityand/or stability, as compared to F1. Variant antibodies or functionalparts thereof comprising an amino acid sequence which is at least 70%identical to a CDR sequence as depicted in FIG. 1 are therefore alsowithin the scope of the present invention. Various methods are availablein the art for altering an amino acid sequence. For instance, a heavychain or light chain sequence with a desired CDR sequence isartificially synthesized. Preferably, a nucleic acid sequence encoding aCDR sequence is mutated, for instance using random—orsite-directed—mutagenesis.

In one embodiment the invention therefore provides an antibody orfunctional part thereof or immunoglobulin chain or functional equivalentthereof, which comprises:

-   -   a heavy chain CDR1 sequence comprising a sequence which has at        least 70% sequence identity to the sequence RFAMS (SEQ ID NO:1),        and/or    -   a heavy chain CDR2 sequence comprising a sequence which has at        least 70% sequence identity to the sequence SINNGNNPYYARSVQY        (SEQ ID NO:2), and/or    -   a heavy chain CDR3 sequence comprising a sequence which has at        least 70% sequence identity to the sequence DHPSSGWPTFDS (SEQ ID        NO:3).

Further provided is an antibody or functional part thereof orimmunoglobulin chain or functional equivalent thereof, which comprises:

-   -   a light chain CDR1 sequence comprising a sequence which has at        least 70% sequence identity to the sequence RASENVGDWLA (SEQ ID        NO:4), and/or    -   a light chain CDR2 sequence comprising a sequence which has at        least 70% sequence identity to the sequence KTSILES (SEQ ID        NO:5), and/or    -   a light chain CDR3 sequence comprising a sequence which has at        least 70% sequence identity to the sequence QHYXRFPYT, wherein X        is I or M (SEQ ID NO:6).

The above mentioned CDR sequences are the CDR sequences of antibody F1;VH IgHV3-23 and VL IgKV1-5, and variants thereof. Binding compoundscomprising CDR sequences with at least 70% sequence identity to F1 CDRsare particularly suitable for counteracting and/or preventing (theeffects of) infections by S. aureus and/or S. epidermidis. It was foundthat a variant of F1, comprising VH IgHV3-23 and VL IgKV1-5, in whichthe isoleucine in light chain CDR3 of the light chain was altered to amethionine, was still capable of specifically binding Staphylococcusspecies such as S. aureus and S. epidermidis.

Preferably, a binding compound according to the invention comprises aCDR sequence which is at least 75%, more preferably at least 80%, morepreferably at least 85%, more preferably at least 86%, more preferablyat least 87%, more preferably at least 88%, more preferably at least89%, more preferably at least 90% identical to at least one of the CDRsequences depicted in FIG. 1. Most preferably, a binding compoundaccording to the invention comprises a CDR sequence which is at least91%, more preferably at least 92%, more preferably at least 93%, morepreferably at least 94%, more preferably at least 95% identical to atleast one of the CDR sequences depicted in FIG. 1. The particularlypreferred antibody F1, described above, comprises CDR sequences whichconsist of the CDR sequences depicted in FIG. 1. A particularlypreferred embodiment according to the invention thus provides anisolated, synthetic or recombinant antibody or a functional equivalentthereof which is capable of specifically binding S. aureus and/or S.epidermidis and which comprises:

-   -   a heavy chain CDR1 sequence comprising the sequence RFAMS (SEQ        ID NO:1), and/or    -   a heavy chain CDR2 sequence comprising the sequence        SINNGNNPYYARSVQY (SEQ ID NO:2), and/or    -   a heavy chain CDR3 sequence comprising the sequence DHPSSGWPTFDS        (SEQ ID NO:3), and/or    -   a light chain CDR1 sequence comprising the sequence RASENVGDWLA        (SEQ ID NO:4), and/or    -   a light chain CDR2 sequence comprising the sequence KTSILES (SEQ        ID NO:5), and/or    -   a light chain CDR3 sequence comprising the sequence QHYXRFPYT,        wherein X is I or M (SEQ ID NO:6).

In one embodiment a binding compound is provided which comprises theheavy chain CDR1 and CDR2 sequences and the light chain CDR1 and CDR2sequences as depicted in FIG. 1, or sequences that are at least 70%,preferably at least 75%, more preferably at least 80%, more preferablyat least 81%, more preferably at least 82%, more preferably at least83%, more preferably at least 84%, more preferably at least 85%identical thereto. Further provided is therefore an isolated, syntheticor recombinant antibody or a functional part thereof or animmunoglobulin chain or a functional equivalent thereof which comprisesa heavy chain CDR1 sequence comprising a sequence which is at least 70%identical to the sequence RFAMS (SEQ ID NO:1) and a heavy chain CDR2sequence comprising a sequence which is at least 70% identical to thesequence SINNGNNPYYARSVQY (SEQ ID NO:2) and a light chain CDR1 sequencecomprising a sequence which is at least 70% identical to the sequenceRASENVGDWLA (SEQ ID NO:4) and a light chain CDR2 sequence comprising asequence which is at least 70% identical to the sequence KTSILES (SEQ IDNO:5). Said binding compound preferably comprises CDR sequences whichare at least 75%, more preferably at least 80%, more preferably at least85%, more preferably at least 86%, more preferably at least 87%, morepreferably at least 88%, more preferably at least 89%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, more preferably at least 93%, more preferably at least 94%, mostpreferably at least 95% identical to the above mentioned heavy chain CDRsequences and light chain CDR sequences. Preferably, said bindingcompound also comprises a heavy chain CDR3 sequence comprising asequence which is at least 70% identical to the sequence DHPSSGWPTFDS(SEQ ID NO:3), and/or a light chain CDR3 sequence comprising a sequencewhich is at least 70% identical to the sequence QHYXRFPYT, wherein X isI or M (SEQ ID NO:6). A binding compound comprising the above mentionedheavy chain CDR1, CDR2 and CDR3 sequences as well as the above mentionedlight chain CDR1, CDR2 and CDR3 sequences is also provided.

Now that a human antibody capable of specifically binding Staphylococcusspecies has been provided by the present invention, it has becomepossible to produce an immunoglobulin chain or functional equivalentthereof comprising at least one CDR sequence of a human immunoglobulinvariable domain which is specific for Staphylococcus species. Furtherprovided is thus an isolated, recombinant or synthetic immunoglobulinchain or functional equivalent thereof comprising at least one CDRsequence of a human immunoglobulin variable region which is specific forStaphylococcus species. In a preferred embodiment, a human antibody isprovided. Optionally, said at least one human CDR sequence or at leastone sequence in at least one of the framework regions is optimized,preferably in order to improve binding efficacy or stability. This isfor instance done by mutagenesis experiments where after the stabilityand/or binding efficacy of the resulting compounds are preferably testedand an improved binding compound is selected.

Besides optimizing CDR sequences in order to improve binding efficacy orstability, it is often advantageous to optimize at least one sequence inat least one of the framework regions. This is preferably done in orderto improve binding efficacy or stability. Framework sequences are forinstance optimized by mutating a nucleic acid molecule encoding suchframework sequence where after the characteristics of the resultingantibody—or functional part—are preferably tested. This way, it ispossible to obtain improved antibodies or functional parts. In apreferred embodiment, human germline sequences are used for frameworkregions in antibodies or functional parts thereof or immunoglobulinchains or functional equivalents according to the invention. The use ofgermline sequences preferably minimizes the risk of immunogenicity ofsaid antibodies, immunoglobulin chains or functional equivalents orparts, because these sequences are less likely to contain somaticalterations which are unique to individuals from which the frameworkregions are derived, and may cause an immunogenic response when appliedto another human individual.

Antibodies or functional parts thereof or immunoglobulin chains orfunctional equivalents thereof comprising a heavy chain amino acidsequence which has at least 70% sequence identity to the heavy chainsequence as depicted in FIG. 1 are also provided. Such heavy chainsequence provides desired binding properties, as evidenced by antibodyF1. Moreover, light chain amino acid sequences which have at least 70%sequence identity to the light chain sequence as depicted in FIG. 1, anda light chain sequence in which an isoleucine in CDR3 is altered tomethionine, also provide desired binding properties, as evidenced byantibody F1, and a variant of antibody F1 comprising said alteration.Further provided is therefore an antibody or functional part orimmunoglobulin chain or functional equivalent according to theinvention, having a heavy chain sequence comprising a sequence which hasat least 70% sequence identity to the sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID NO:7)and/or having a light chain sequence which has at least 70% sequenceidentity to the sequenceDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRTV, wherein inX is I or M (SEQ ID NO:8).

Several variants of the F1 antibody have been developed, besides thevariant indicated herein above in which an isoleucine in light chainCDR3 is altered to methionine. These variants are capable of binding toStaphylococcus species. Examples of such antibody variants includeantibodies or functional parts or immunoglobulin chains or functionalequivalents according to the invention, having a heavy chain sequencecomprising the sequenceEVQLVESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ IDNO:9), and/or the sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID NO:7),and a light chain sequence comprising the sequenceDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRA, wherein X is I of M (SEQID NO:10), and/or the sequence DIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKVEIK RTV,wherein X is I of M (SEQ ID NO:11), and/or the sequenceDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRTV, wherein X is I of M (SEQID NO:8).

An antibody or functional part or immunoglobulin chain or functionalequivalent thereof according to the invention specifically binds toproteins comprising a serine-aspartate (SD) repeat. SD repeat (Sdr)proteins are cell-surface-associated proteins that are present inseveral bacteria such as Staphylococcus species. Sdr proteins in generalcomprise an amino-terminal signal sequence, a functional domain calledthe A region, an SD repeat region, a cell wall-spanning region, a LPXTGmotif, a hydrophobic membrane-spanning domain, and a series ofpositively charged residues. The LPXTG motif is the target of atranspeptidase that cleaves the motif between threonine and glycineresidues and anchors the protein to the peptidoglycan of the cell wallof gram-positive bacteria. Sdr proteins are thought to interact withhost molecules. Known Sdr proteins include ClfA (SdrA), ClfB (SdrB),SdrC, SdrD and SdrE of S. aureus, SdrF, SdrG and SdrH of S. epidermidis,SdrI of S. saprophyticus, SdrX of S. capitis, and SdrY and SdrZ of S.caprae.

Therefore, a preferred antibody or functional part thereof orimmunoglobulin chain or functional equivalent according to the inventionspecifically binds S. aureus, S. epidermidis, S. saprophyticus, S.capitis, and S. caprae. It is preferred that said antibody,immunoglobulin chain or functional equivalent or part thereof binds toClfA (SdrA), ClfB (SdrB), SdrC, SdrD and SdrE of S. aureus, SdrF, SdrGand SdrH of S. epidermidis, SdrI of S. saprophyticus, SdrX of S.capitis, and SdrY and SdrZ of S. caprae. The epitope of the antibody,immunoglobulin chain or functional equivalent or part thereof accordingto the invention comprises an SD repeat-dependent epitope in Sdrproteins, for instance SD repeat-dependent epitopes as present in S.aureus ClfA, ClfB, SdrC, SdrD and SdrE. An SD repeat-dependent epitopeis herein defined as an epitope recognized by antibody F1, which epitoperequire the presence of at least part of an SD repeat region as presentin, but not limited to, S. aureus ClfA, ClfB, SdrC, SdrD and SdrE and S.epidermidis SdrF, SdrG and SdrH. In one embodiment said epitope maycomprise at least part of a molecule that binds to, or is associatedwith an Sdr protein. Examples of such molecules include, but are notlimited to, amino acids, peptides, proteins, sugars and sugar residues.In another embodiment, said epitope comprises modifications of the SDrepeat region. Said modifications comprise, for example, but are notlimited to, glycosylation, amidation and/or phosphorylation. It will beclear to a skilled person that combination of these two embodiments isalso possible.

Therefore, the invention provides an antibody or functional part orimmunoglobulin chain or functional equivalent according to the inventioncapable of binding an SD repeat-dependent epitope. Also provided is anantibody or functional part or immunoglobulin chain or functionalequivalent capable of binding to S. aureus ClfA, ClfB, SdrC, SdrD andSdrE. Further provided is an antibody or functional part orimmunoglobulin chain or functional equivalent which is capable ofcompeting with an antibody or functional part or immunoglobulin chain orfunctional equivalent according to the present invention for binding toa Staphylococcus species, preferably S. aureus and/or S. epidermidisand/or S. saprophyticus and/or S. capitis and/or S. caprae, morepreferably MRSA.

A disadvantage of antibodies is that their stability may be reduced, forexample under harsh conditions. For instance deamidation, the removal ofa functional amide group, may occur. Deamidation is a proteindegradation pathway that may affect the biological functions ofproteins, and which occurs mainly at asparagine residues, and to a lowerextent at glutamine residues. In one embodiment, therefore, deamidationof an antibody or functional part thereof or immunoglobulin chain orfunctional equivalent thereof according to the invention is prevented byreplacing an asparagine or glutamine by another amino acid. Anasparagine is preferably replaced by an amino acid other than glutamine,because deamidation may also occur at a glutamine residue. Replacementof an asparagine preferably does not substantially affect bindingaffinity of an antibody according to the invention to an antigen. In oneembodiment deamidation of an asparagine at position 53 of the heavychain (numbering according to Kabat, 1991) is prevented by replacingsaid asparagine by another amino acid. By preventing deamidation of anasparagine at position 53 the stability of an antibody or functionalpart thereof or immunoglobulin chain or functional equivalent thereofaccording to the invention is preferably increased. As is shown in theexamples, despite the fact that said asparagine is located in a CDR,replacing an asparagine at said position does not substantially affectthe binding affinity for an antigen of an antibody or functional partthereof or immunoglobulin chain or functional equivalent thereofaccording to the invention. An asparagine at position 53 of the heavychain is preferably replaced by an amino acid other than glutamine, morepreferably an asparagine at said position is replaced by serine.Therefore, also provided by the invention is therefore an antibody orfunctional part or immunoglobulin chain or functional equivalentaccording to the invention, wherein an asparagine, preferably anasparagine at position 53 of the heavy chain, is replaced by anotheramino acid, preferably serine.

In one embodiment, an antibody or functional part thereof orimmunoglobulin chain or functional equivalent thereof according to theinvention is coupled to another moiety to form antibody-drug conjugates.An antibody or functional part thereof or immunoglobulin chain orfunctional equivalent thereof according to the invention is for instancecoupled to a cytotoxic agent, such as an antibiotic. The term “cytotoxicagent” as used herein refers to a substance that reduces or blocks thefunction, or growth, of bacteria and/or causes destruction of bacteria.Said other moiety, for example a cytotoxic agent, is preferably coupledto said antibody or functional part thereof through a thiol group.Therefore, preferably one or more cysteines are incorporated into saidantibody or functional part thereof or immunoglobulin chain orfunctional equivalent thereof. Cysteines contain a thiol group andtherefore, incorporation of one or more cysteines into, or replacementof one or more amino acids by one or more cysteines of an antibody orfunctional part thereof according to the invention enable couplingthereof to another moiety. Said one or more cysteines are preferablyintroduced into an antibody of functional equivalent thereof accordingto the invention at a position where it does not influence folding ofsaid antibody or functional equivalent, and does not alter antigenbinding or effector function. The invention therefore provides anantibody or functional part thereof or immunoglobulin chain orfunctional equivalent thereof according to the invention wherein atleast one amino acid other than cysteine has been replaced by acysteine. Preferably at least two amino acids other than cysteine havebeen replaced by cysteine. In a preferred embodiment, said at least oneamino acid other than cysteine is valine at light chain position 15,and/or alanine at light chain position 144, and/or serine at light chainposition 168, and/or valine at light chain position 205 and/or valine atlight chain position 110, and/or alanine at heavy chain position 84,and/or alanine at heavy chain position 114, and/or alanine at heavychain position 168, and/or serine at heavy chain position 172, morepreferably valine at light chain position 205 and/or valine at lightchain position 110, and/or alanine at heavy chain position 114(numbering according to Kabat, 1991). A skilled person will understandthat, as an alternative or in addition, one or more other amino acids ofthe heavy and/or light chain can be replaced by cysteine if thereplacement does not influence folding of said antibody or functionalequivalent, and does not alter antigen binding or effector function.

In international patent applications WO2006/034488, WO2008/141044,WO2009/052249, WO2009/012256, WO2009/012268 and WO2009/099728 methodsfor engineering antibodies with reactive cysteine residues are describedas well as amino acid positions suitable for cysteine engineering.

An antibody or functional part or immunoglobulin chain or functionalequivalent according to the invention preferably comprises a variableheavy chain sequence and/or a variable light chain sequence which is atleast 75%, more preferably at least 80%, more preferably at least 85%,more preferably at least 90%, most preferably at least 95% identical toa heavy chain sequence and/or the light chain sequence as depicted inFIG. 1, or a light chain sequence as depicted in FIG. 1 in which theisoleucine in CDR3 is altered to a methionine. The higher the identity,the more closely said binding compound resembles antibody F1. Anantibody or functional part or immunoglobulin chain or functionalequivalent according to the invention preferably comprises a heavy chainas well as a light chain which resemble the heavy and light chain of F1.Further provided is therefore an antibody or functional part thereof orimmunoglobulin chain or functional equivalent thereof comprising a heavychain sequence and a light chain sequence which are at least 70%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, most preferably at least 95% identical to the heavy chainsequence and the light chain sequence as depicted in FIG. 1, or a lightchain sequence as depicted in FIG. 1 in which the isoleucine in CDR3 isaltered to a methionine. In one embodiment an antibody or functionalpart is provided which has a heavy chain sequence as depicted in FIG. 1and a light chain sequence as depicted in FIG. 1 or a light chainsequence as depicted in FIG. 1 in which the isoleucine in CDR3 isaltered to a methionine.

One embodiment provides an antibody or functional part thereof orimmunoglobulin chain or functional equivalent thereof comprising a heavychain sequence consisting of the heavy chain sequence as depicted inFIG. 1, and/or comprising a light chain sequence consisting of the lightchain sequence as depicted in FIG. 1 or a light chain sequence asdepicted in FIG. 1 in which the isoleucine in CDR3 is altered to amethionine. Alternatively, as is well known by the skilled person, it ispossible to generate a shortened heavy chain or light chain sequencewhile maintaining a binding property of interest. Preferably, such ashortened heavy chain or light chain is generated which has a shorterconstant region, as compared to the original heavy or light chain. Thevariable domain is preferably maintained. For instance, a Fab fragmentor F(ab′)₂ fragment or a single domain antibody or a single chainantibody or a nanobody or an unibody or a scFv fragment based on a heavychain sequence or light chain sequence depicted in FIG. 1 is produced. Afunctional part of an antibody comprising at least a functional part ofa sequence as depicted in FIG. 1, or a light chain sequence as depictedin FIG. 1 in which the isoleucine in CDR3 is altered to a methionine, istherefore also provided. Said functional part has a length of at least20 amino acids and comprises at least one sequence selected from thegroup consisting of a sequence which is at least 70% identical to theheavy chain CDR1 sequence depicted in FIG. 1 and a sequence which is atleast 70% identical to the heavy chain CDR2 sequence depicted in FIG. 1and a sequence which is at least 70% identical to the heavy chain CDR3sequence depicted in FIG. 1 and a sequence which is at least 70%identical to the light chain CDR1 sequence depicted in FIG. 1 and asequence which is at least 70% identical to the light chain CDR2sequence depicted in FIG. 1 and a sequence which is at least 70%identical to the light chain CDR3 sequence depicted in FIG. 1, or alight chain CDR3 sequence as depicted in FIG. 1 in which the isoleucineis altered to a methionine.

The invention further provides an isolated, synthetic or recombinantnucleic acid sequence or a functional equivalent thereof with a lengthof at least 15 nucleotides, preferably at least 30 nucleotides, morepreferably at least 50 nucleotides, more preferably at least 75nucleotides, encoding a binding compound according to the invention.Such nucleic acid is for instance isolated from a B-cell which iscapable of producing an antibody according to the invention. A preferredembodiment provides a nucleic acid sequence comprising a sequence whichhas at least 70% sequence identity to at least 15 nucleotides of anucleic acid sequence as depicted in FIG. 1. A nucleic acid sequenceaccording to the invention preferably comprises a sequence which has atleast 75%, more preferably at least 80%, more preferably at least 85%,more preferably at least 90%, most preferably at least 95% sequenceidentity to at least 15 nucleotides of a nucleic acid sequence asdepicted in FIG. 1. Preferably, said nucleic acid sequence as depictedin FIG. 1 comprises at least one CDR encoding sequence.

One preferred embodiment provides an isolated, synthetic or recombinantnucleic acid sequence with a length of at least 15 nucleotides, or afunctional equivalent thereof, encoding at least one CDR sequence of anantibody or immunoglobulin chain according to the invention. Saidnucleic acid sequence preferably encodes at least one CDR sequence whichhas at least 70% sequence identity to a CDR region of antibody F1.Nucleic acid sequences encoding F1 CDR regions are depicted in FIG. 1.Further provided is therefore an isolated, synthetic or recombinantnucleic acid sequence, or a functional equivalent thereof, comprising asequence which has at least 70% sequence identity to a sequence selectedfrom the group consisting of cgctttgccatgagc (SEQ ID NO:12),tcgatcaataatgggaataacccatactacgcacggtcggtacaatac (SEQ ID NO:13),gatcaccctagtagtggctggcccacctttgactcc (SEQ ID NO:14),cgggccagtgaaaacgttggtgactggttggcc (SEQ ID NO:15), aagacatctattctagaaagt(SEQ ID NO:16) and caacactatatacgtttcccgtacact (SEQ ID NO:17).

Said nucleic acid sequence or functional equivalent preferably comprisesa sequence which has at least 75%, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90% sequence identityto any of the above mentioned sequences. Further provided is a nucleicacid sequence or functional equivalent thereof comprising a sequencewhich has at least 70% sequence identity to at least part of anucleotide sequence as depicted in FIG. 1, said part having at least 15nucleotides and encoding at least one CDR region.

A nucleic acid sequence or functional equivalent thereof according tothe present invention preferably encodes a region which has at least 70%sequence identity to an F1 CDR region, an F1 heavy chain and/or an F1light chain. One embodiment thus provides an isolated, synthetic orrecombinant nucleic acid sequence, or a functional equivalent thereof,comprising a sequence encoding an amino acid sequence which has at least70% sequence identity to the sequence RFAMS (SEQ ID NO:1), and/or atleast 70% sequence identity to the sequence SINNGNNPYYARSVQY (SEQ IDNO:2), and/or at least 70% sequence identity to the sequenceDHPSSGWPTFDS (SEQ ID NO:3), and/or at least 70% sequence identity to thesequence RASENVGDWLA (SEQ ID NO:4), and/or at least 70% sequenceidentity to the sequence KTSILES (SEQ ID NO:5), and/or at least 70%sequence identity to the sequence QHYXRFPYT, wherein X is I or M (SEQ IDNO:6), and/or at least 70% sequence identity to the sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID NO:7), and/or at least 70% sequence identity to thesequenceDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRTV, wherein X is I or M (SEQID NO:8). Also provided are nucleic acid sequences or functionalequivalents thereof encoding variants of the F1 antibody according tothe invention. Provided by the invention are, for example, nucleic acidsequences encoding a heavy chain sequence comprising the sequenceEVQLVESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID NO:9), and/or thesequence EVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID NO:7),and encoding a light chain sequence comprising the sequenceDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRA, wherein X is I of M (SEQ ID NO:10), and/or thesequenceDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKVEIKRTV, wherein X is I of M (SEQID NO:11), and/or the sequence DIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEI KRTV,wherein X is I of M (SEQ ID NO:8).

In one embodiment an asparagine at position 53 of the heavy chain(numbering according to Kabat, 1991) is replaced by another amino acidother than glutamine, in order to prevent deamidation of the asparagineat said position. Preferably said asparagine is replaced by a serine.

The term “% sequence identity” is defined herein as the percentage ofresidues in a candidate amino acid of nucleic acid sequence that isidentical with the residues in a reference sequence after aligning thetwo sequences and introducing gaps, if necessary, to achieve the maximumpercent identity. Methods and computer programs for the alignment arewell known in the art.

As used herein, a nucleic acid molecule or nucleic acid sequence of theinvention preferably comprises a chain of nucleotides, more preferablyDNA and/or RNA. In other embodiments a nucleic acid molecule or nucleicacid sequence of the invention comprises other kinds of nucleic acidstructures such as for instance a DNA/RNA helix, peptide nucleic acid(PNA), locked nucleic acid (LNA) and/or a ribozyme. Such other nucleicacid structures are referred to as functional equivalents of a nucleicacid sequence. The term “functional equivalent of a nucleic acidsequence” also encompasses a chain comprising non-natural nucleotides,modified nucleotides and/or non-nucleotide building blocks which exhibitthe same function as natural nucleotides.

A nucleic acid sequence according to the present invention isparticularly useful for generating binding compounds which are specificfor S. aureus and/or S. epidermidis. This is for instance done byintroducing such nucleic acid sequence into a cell so that the cell'snucleic acid translation machinery will produce the encoded bindingcompound. In one embodiment, genes encoding a heavy and/or light chainaccording to the invention are expressed in so called producer cells,such as for instance cells of a Chinese hamster ovary (CHO), NSO (amouse myeloma) or 293(T) cell line, some of which are adapted tocommercial antibody production. Proliferation of said producer cellsresults in a producer cell line capable of producing antibodies orfunctional parts thereof according to the present invention. Preferably,said producer cell line is suitable for producing compounds for use inhumans. Hence, said producer cell line is preferably free of pathogenicagents such as pathogenic micro-organisms. Most preferably, bindingcompounds consisting of human sequences are generated using a nucleicacid sequence according to the invention.

An isolated or recombinant antibody producing cell capable of producingan antibody or functional part or immunoglobulin chain or functionalequivalent according to the invention is therefore also provided, aswell as a method for producing an antibody or functional part orimmunoglobulin chain or functional equivalent according to theinvention, comprising providing a cell with a nucleic acid sequence orfunctional equivalent according to the invention and allowing said cellto translate said nucleic acid sequence or functional equivalentaccording to the invention, thereby producing said antibody orfunctional part or immunoglobulin chain or functional equivalentaccording to the invention.

An antibody producing cell is defined herein as a cell which is capableof producing and/or secreting an antibody or a functional part thereof,and/or which is capable of developing into a cell which is capable ofproducing and/or secreting antibody or a functional part thereof. Anantibody producing cell according to the invention is preferably aproducer cell which is adapted to commercial antibody production.Preferably, said producer cell is suitable for producing compounds foruse in humans.

A method according to the invention preferably further comprises a stepof harvesting, purifying and/or isolating said antibody or functionalpart or immunoglobulin chain or functional equivalent according to theinvention. The thus obtained binding compounds according to theinvention are preferably used in diagnosis of Staphylococcus infection,isolation or detection of Staphylococcus bacteria, distinguishingbetween Staphylococcus species and other gram-positive bacteria andhuman therapy, optionally after additional purification, isolationand/or other processing steps.

An antibody or functional part thereof or immunoglobulin chain orfunctional equivalent thereof according to the present invention isparticularly suitable for diagnostic uses. For instance, a sample, suchas a tissue or blood sample, can be obtained from an individual or fromany other source suspected to be infected with a Staphylococcusbacteria, preferably S. aureus and/or S. epidermidis, more preferablyMRSA. Subsequently, said sample can be mixed with an antibody orimmunoglobulin chain or functional equivalent or part thereof accordingto the invention. Said antibody, immunoglobulin chain or functionalequivalent or part will specifically bind to Staphylococcus bacteria,preferably S. aureus and/or S. epidermidis. Bacteria bound to anantibody, immunoglobulin chain or functional equivalent or part can beisolated from the sample using any method known in the art, for example,but not limited to, isolation using magnetic beads, streptavidin-coatedbeads, or isolation through the use of secondary antibodies immobilizedon a column. After washing of the bound bacteria and antibody,immunoglobulin chain or functional equivalent or part thereof, bacteriacan be eluted from said antibody, immunoglobulin chain or functionalequivalent or part by any method known in the art. For instance, bindingbetween bacteria and antibody, immunoglobulin chain or functionalequivalent or part can be disrupted by increasing the concentration ofsalts and/or reducing or increasing pH, and/or by addition of excessepitope.

Isolation of Staphylococcus bacteria, preferably S. aureus and/or S.epidermidis, may be used for various applications. For instance,infection with several different gram-positive bacteria may result inoverlapping symptoms in an individual. In such cases, it can bedifficult to discriminate between S. aureus and/or S. epidermidis andother gram-positive bacteria. An antibody, immunoglobulin chain orfunctional equivalent or part thereof according to the present inventioncan then be used to detect the presence of S. aureus and/or S.epidermidis, or for distinguishing between S. aureus and/or S.epidermidis and other bacteria. Isolation of bacteria from a sample ofan individual suspected of suffering from an infection with S. aureusand/or S. epidermidis or from any other source, such as a bacterialculture, can facilitate detection of said Staphylococcus bacteria,because isolation results in an increased concentration and/or a higherpurity of said Staphylococcus bacteria.

Isolation of Staphylococcus species, preferably S. aureus and/or S.epidermidis, more preferably MRSA, can for instance further be used toidentify the specific S. aureus and/or S. epidermidis strain, preferablythe MRSA strain in a sample. Identification of said strain can forinstance be performed by determining the sequence of the bacterial DNA.In such case it is preferred to first obtain an isolated S. aureusand/or S. epidermidis.

In one embodiment of the invention, an antibody, immunoglobulin chain orfunctional equivalent or part thereof according to the present inventionis labeled in order to be able to detect said antibody, immunoglobulinchain, or functional equivalent or part, for instance, but not limitedto, fluorescently labeled, or radioactively labeled. Alternatively, anantibody or functional part thereof or immunoglobulin chain orfunctional equivalent thereof according to the invention is detectedusing a labeled secondary antibody which is directed against saidantibody, immunoglobulin chain or functional equivalent or part thereofaccording to the invention. If binding of said antibody, immunoglobulinchain or functional equivalent or part thereof is detected, S. aureusand/or S. epidermidis is present.

Provided by the invention is therefore a use of an antibody orfunctional part thereof or immunoglobulin chain or functional equivalentthereof according to the invention for diagnosis of a Staphylococcusinfection, preferably S. aureus infection, more preferably MRSAinfection, for detecting S. aureus and/or S. epidermidis, preferablyMRSA, and for distinguishing between S. aureus and/or S. epidermidis,preferably MRSA and other gram-positive bacteria. Also provided is anantibody or functional part thereof or an immunoglobulin chain orfunctional equivalent thereof according to the invention for use in thediagnosis of a Staphylococcus infection, preferably S. aureus infection,more preferably MRSA infection.

Further provided is a method for isolating S. aureus and/or S.epidermidis bacteria from a solution using an antibody or functionalpart or an immunoglobulin chain or functional equivalent thereofaccording to the invention. Said method preferably comprises providing asample of an individual suspected of suffering from an infection with S.aureus and/or S. epidermidis, preferably MRSA, or from any other source,such as a bacterial culture, adding an antibody or functional part or animmunoglobulin chain or functional equivalent thereof according to theinvention to said sample, allowing binding of said antibody orfunctional part or an immunoglobulin chain or functional equivalentthereof according to the invention to S. aureus and/or S. epidermidisbacteria, when present, and isolating S. aureus and/or S. epidermidisbacteria bound to an antibody or functional part or an immunoglobulinchain or functional equivalent thereof according to the invention fromsaid sample.

Now that Gram-positive bacteria-specific binding compounds according tothe invention and nucleic acid sequences coding therefore have beenprovided, including human binding compounds, improved therapeuticapplications have become available. Gram-positive bacteria such as S.aureus and/or S. epidermidis are counteracted by binding compoundsaccording to the invention. A binding compound according to theinvention is therefore particularly suitable for use as a medicine orprophylactic agent. Preferably, binding compounds are used which consistof human sequences, or which have at most 5% of non-human sequences, inorder to reduce the chance of adverse side effects when humanindividuals are treated. An antibody or functional part or animmunoglobulin chain or functional equivalent thereof or a nucleic acidsequence or functional equivalent thereof according to the invention foruse as a medicament and/or prophylactic agent is therefore also herewithprovided. When a nucleic acid or functional equivalent according to theinvention is administered, it will be translated in situ into a bindingcompound according to the invention. In a particularly preferredembodiment said antibody comprises antibody F1, or a functional partthereof. Said medicament or prophylactic agent is preferably used forcounteracting or at least in part preventing an infection by S. aureusand/or S. epidermidis or for counteracting or at least in partpreventing adverse effects of an infection by S. aureus and/or S.epidermidis. Further provided is therefore an antibody or functionalpart or an immunoglobulin chain or functional equivalent thereof or anucleic acid sequence or functional equivalent thereof according to theinvention for use as a medicament and/or prophylactic agent for at leastin part treating and/or preventing a condition related to S. aureusand/or S. epidermidis. Non-limiting examples of such conditions are skininfections, pimples, impetigo, boils, cellulitis folliculitis,furuncles, carbuncles, scalded skin syndrome, abscesses, pneumonia,meningitis, osteomyelitis, endocarditis, toxic shock syndrome andsepticemia. Preferably, S. aureus infection is counteracted or at leastin part prevented. Most preferably, an MRSA-related condition iscounteracted, diminished and/or at least in part prevented. A use of anantibody or functional part or an immunoglobulin chain or functionalequivalent thereof or a nucleic acid sequence or functional equivalentthereof according to the invention for the preparation of a medicamentand/or prophylactic agent for at least in part treating and/orpreventing S. aureus and/or S. epidermidis is therefore also provided,as well as a method for at least in part treating or preventing acondition related to S. aureus and/or S. epidermidis, the methodcomprising administering to an individual in need thereof atherapeutically effective amount of an antibody or functional part or animmunoglobulin chain or functional equivalent according to theinvention, preferably after said individual has been diagnosed to beinfected by S. aureus and/or S. epidermidis. Said condition preferablycomprises at least one of the S. aureus-related conditions listed above,most preferably an MRSA-related condition.

Said antibody preferably comprises antibody F1, or a functional partthereof.

In order to counteract Gram-positive bacteria, a binding compoundaccording to the invention is preferably administered to an individualbefore an infection has taken place. Alternatively, a binding compoundaccording to the invention is administered when an individual is alreadyinfected. Said binding compound is preferably administered toindividuals with an increased risk of complications, such as forinstance hospitalized individuals and/or individuals with compromisedimmunity. Also elderly people have an increased risk of bacterialconditions. Binding compounds according to the invention are preferablyadministered via one or more injections. Dose ranges of bindingcompounds according to the invention to be used in the therapeuticapplications as described herein before are designed on the basis ofrising dose studies in the clinic in clinical trials for which rigorousprotocol requirements exist. Typical doses are between 0.1 and 10 mg perkg body weight. For therapeutic application binding compounds accordingto the invention are typically combined with a pharmaceuticallyacceptable carrier, diluent and/or excipient. Examples of suitablecarriers for instance comprise keyhole limpet haemocyanin (KLH), serumalbumin (e.g. BSA or RSA) and ovalbumin. In one preferred embodimentsaid suitable carrier comprises a solution like for example saline.

In yet another embodiment a nucleic acid encoding a binding compoundaccording to the invention is used. As already described, uponadministration of such nucleic acid, binding compounds are produced bythe host's machinery. Produced binding compounds are capable ofpreventing and/or counteracting Gram-positive bacterial infection and/orthe adverse effects of such infection. A nucleic acid sequence orfunctional equivalent according to the invention for use as a medicamentand/or prophylactic agent is therefore also herewith provided. Saidnucleic acid is preferably used for counteracting S. aureus and/or S.epidermidis, more preferably S. aureus, most preferably MRSA. Furtherprovided is therefore a use of a nucleic acid sequence or functionalequivalent according to the invention for the preparation of amedicament and/or prophylactic agent for at least in part treatingand/or preventing a Gram-positive bacterium-related condition. SaidGram-positive bacterium-related condition preferably comprises aninfection by S. aureus or S. epidermidis, more preferably an S. aureusinfection, most preferably an MRSA infection.

Further provided is a pharmaceutical composition comprising an antibodyor functional part or an immunoglobulin chain or functional equivalentthereof or a nucleic acid sequence or functional equivalent thereofaccording to the invention and a pharmaceutical acceptable carrier,diluent or excipient. Said pharmaceutical composition is preferablysuitable for human use.

The invention is further explained in the following examples. Theseexamples do not limit the scope of the invention, but merely serve toclarify the invention.

All patent documents and other publications that are referred to in thedetailed description are incorporated herein by reference in theirentirety.

EXAMPLES Example 1 Methods B Cell Isolation

B-cells were obtained from fresh blood of adults by Ficoll separationand CD4/CD8 negative selection with MACS microbeads as described by themanufacturer (Miltenyi Biotech). To obtain memory B cells, cells weresorted for CD19⁺CD3⁻CD27⁺IgD⁻IgA⁻ on a FACSaria (Becton Dickinson). Theuse of these tissues was approved by the medical ethical committees ofthe institution and was contingent on informed consent. Donors wereselected based on carrying hospital acquired MRSA strains with genotypecluster 109 and 16.

Cell Culture

B-cells maintained in standard culture medium containing IMDM (Gibco),8% FBS (HyClone) and penicillin/streptomycin (Roche) were co-cultured onγ-irradiated (50Gy) mouse L cell fibroblasts stably expressing CD40L(CD40L-L cells, 10⁵ cells/ml) and recombinant mouse IL-21 (25 ng/ml, R&Dsystems). rhIL-4 (R&D) was used at 50 ng/ml. Cells were routinely testedby PCR and were found negative for the presence of mycoplasma and EBV(data not shown).

Bulk transduced human memory B cells double positive for NGFR and GFPwere purified by FACS cell sorting and cultured in 96 well plates at acell density of 500 cell per well. Culture supernatants were tested inELISAs using bacterial lysates of the Newman and SH1000 strains.Positive cultures were subcloned at 10 cell/well cell density in 96 wellplates and again tested by ELISA. Subsequently positive cultures wereseeded at 1 cell per well and tested again by ELISA for reactivityagainst S. aureus strains Newman and SH1000.

Retroviral Transduction

The BCL6 retroviral construct has been described before (Shvarts A. etal. Genes Dev 16, 681-686 (2002)). cDNA encoding human Bcl-xL was kindlyprovided by Dr. Stanley Korsmeyer. BCL6 and Bcl-xL were separatelycloned into a BCL6-NGFR and a BclxL-GFP construct. These constructs weretransfected into the LZRS retroviral packaging cells, phoenix asdescribed before (Jaleco A. C. et al. Blood 94, 2637-2646 (1999);Scheeren F. A. et al. Nat Immunol 6, 303-313 (2005)). Memory B-cellswere double transduced by the BCL6 and Bcl-xL containing retrovirusesafter activation on CD40L-L cells in the presence of rmIL-21 for 36 hrsas described before (Diehl S. A. et al. J Immunol 180, 4805-4815 (2008);Kwakkenbos M. et al. Nat Med in press (2009)). Transduced cells weremaintained on CD40L-L cells with rmIL-21.

Elisa

To determine antibody content ELISA plates were coated with anti-humanIgG (Jackson ImmunoResearch Laboratories) at 5 μg/ml in PBS for 1 hr at37° C. or o/n at 4° C. and washed in ELISA wash buffer (PBS, 0.5%Tween-20). 4% milk in PBS was used as blocking agent, before serialdilution of cell culture supernatants and an enzyme-conjugated detectionantibody was added (dilution 1:2500 for HRP-conjugated anti-IgG(Jackson)). TMB substrate/stop solution (Biosource) was used fordevelopment of the ELISAs.

For screening purposes we used lysates from the Newman and SH1000strains. Both were made in PBS and directly coated at 5 to 10 μg ml⁻¹before B cell culture supernatants were tested un- or 1:2 diluted.

To explore the antigen specificity of the human IgG1 clone F1, an LTAdetection ELISA was developed. Purified LTA preparations from S. aureus,B. subtilis, S. faecalis and S. pyogenes (Sigma) was added to ELISAplates coated with polyclonal IgG purified mouse anti-LTA (1/200 stock 1mg ml⁻¹, QED Bioscience), before secondary antibodies were added.

In addition, we tested several LTA preparations derived from a librarydeveloped by W. Fischer (Institut fur Biochemie, Univ of Erlangen,Germany) and kindly provided by B. Appelmelk (VU, Amsterdam,Netherlands) (see for more details (Keller R. et al. Infect Immun 60,3664-3672 (1992), Polotsky V. Y. et al. Infection and Immunity 64,380-383 (1996) and Greenberg J. W. et al. Infection and Immunity 64,3318-3325 (1996)) and reviewed in (Weidenmaier C. et al. Nat RevMicrobiol 6, 276-287 (2008)) in direct ELISA's. LTA preparations werecoated at 1 μg ml⁻¹ before rF1 (10 μg ml⁻¹) or control antibodies (1:5dilution of hybridoma supernatant) were added and further detected withconjugated anti-human or mouse antibodies. The panel of purified LTApreparations included: B. subtilis, S. aureus, L. lactis, L. garvieae,B. bifidum, M. luteus, L. casei, L. mesenteroides, B. licheniformis, L.welshimeri, E. hirae, L. raffinolactis, S. mutans, S. pneumoniae.Several variants contain or lack alanine residues and/or lipid anchors(not depicted here).

Binding of the F1 Antibody to Bacterial Cultures

The Newman S. aureus and the S. pneumoniae strain (serotype 3) wereused. Newman was cultured in TSH 50 ml O/N, then 1 ml was resuspendendin 100 ml for 2 to 2.5 hrs till OD:1 and bacteria were collected. S.pneumoniae was cultured in Todd Hewitt medium mixed with yeast medium.Before, bacteria were incubated with F1 antibody, a control IgG (D25, ahuman anti-RSV antibody) or only with the secondary antibody (IgG-PEonly), cells were pretreated with 100% total mouse serum to preventbackground staining. After washing the secondary antibody was added(IgG-PE). Antibody incubations were performed for 20 min on ice.

F1 Sequence Determination and Expression Cloning

We isolated total RNA using Trizol (Invitrogen), generated cDNA by usingsuperscript RT, performed PCR and cloned the heavy and light chainvariable regions into the pCR2.1 TA cloning vector (Invitrogen). To ruleout reverse transcriptase or DNA polymerase induced mutations, weperformed several independent cloning experiments. To producerecombinant F1 mAb we cloned F1 heavy and light variable regions inframe with human IgG1 and Kappa constant regions into a pcDNA3.1(Invitrogen) based vector and transiently transfected 293T cells. Wepurified recombinant F1 from the culture supernatant with Protein A.

Results Generation of the F1 Clone

From three subjects who were tested positive for MRSA but were not sick,50-60 ml of heparin blood was collected and peripheral B-cells wereisolated after a ficoll purification step. B-cells from several B-cellpopulations were double transduced with retroviruses containingBCL6-NGFR and Bcl-xL-GFP (Diehl et al and Kwakkenbos et al). From theIgG, CD27 positive population, polyclonal mini-cultures B-cell cultureswere started in 96 wells plate with a cell density of 500 cells/well.Supernatant of these mini-cultures was collected and used in ELISAs toscreen for the presence of S. aureus-specific IgG antibodies (in theseELISAs the coating was cell lysates of two S. aureus strains, SH1000 andNewman). Mini-cultures that were screened positive in both the SH1000 aswell as the Newman S. aureus were seeded in new mini-cultures at adensity of 10 cells/well. Again, supernatant of these mini-cultures wascollected and screened in the SH1000 and Newman ELISAs. Clones that werescreened positive in both ELISAs were seeded into monoclonal cultures(i.e. 1 cell/well). After testing the supernatant of these monoclonalcultures, one clone (named F1 ) was found to produce S. aureus specificmonoclonal IgG antibodies.

Antibodies in the Supernatant of the F1 Clone Bind to LTA Preparationsfrom S. aureus

Generating the F1 clone, we already found that supernatant of F1 boundto the bacterial cell lysates of two S. aureus strains, SH1000 andNewman. The major cell wall compound of Gram-positive bacteria is LTA,and therefore we decided to test binding of F1 supernatant to S. aureusLTA preparations in an ELISA. As shown in Table 1, supernatant of the F1clone binds to bacterial cell lysates of the SH1000 and Newman S. aureusstrain but also to commercially purchased purified S. aureus LTApreparations. We noted, however, that the binding to the LTApreparations was significantly lower than that observed with wholebacteria.

TABLE 1 Supernatant of the F1 clone binds to S. aureus LTA preparations.As a negative control an anti-mouse-HRP conjugated secondary antibodywas used. Another control was an anti-influenza virus antibody that onlydetected influenza H3 protein. mouse anti-flu F1 clone anti-LTA controlSH1000 1.004 −0.010 −0.007 Newman 0.753 −0.007 0.007 SA LTA (sigma)0.176 −0.005 −0.009 Flu H3 0.003 −0.002 1.523 No coat −0.013 −0.014−0.015The Recombinant Produced F1 Antibody Binds to LTA Preparations fromMultiple Sources

After cloning the antibody genes into expression vectors and producingrecombinant F1 (rF1) antibody in an expression system, the rF1 antibodywas tested on purified LTA preparations from several bacteria. As shownin FIG. 2A, the rF1 antibody binds well to commercial LTA samplesobtained from S. aureus and B. subtilis, and a bit less profoundly toLTA samples from S. faecalis and S. pyogenes. The rF1 antibody did notbind to an LTA sample from S. pneumoniae (data not shown). In additionrF1 recognized highly purified LTA samples from B. subtilis, B.licheniformis and two isolates of S. aureus (FIG. 2 b). rF1 did not bindto LTA preparations from S. mutans (FIG. 2 b) or from L. lactis, L.garvieae, B. bifidum, M. luteus, L. casei, L. mesenteroides, L.welshimeri, E. hirae, L. raffinolactis, S. mutans and S. pneumoniae(results not shown).

Recombinant F1 Antibody Binds to Living S. aureus Bacteria

In order to study whether the rF1 antibody would also recognize livingGram-positive bacteria, binding of the rF1 antibody to S. aureus and S.pneumoniae was tested by using flow cytometry. As shown in FIG. 3A, therF1 antibody binds to living S. aureus bacteria (Newman strain), but notto S. pneumoniae. In addition we show that rF1 recognized 6 clinical S.aureus isolates; one is a pathogenic strain which is PVL positive, 3regular strains and 2 MRSA strain (FIG. 3 b).

Example 2 Methods Bacterial Strains and Culture

Methicillin-resistant S. aureus (MRSA) strains USA300 (1114), USA400,N315, USA100, USA1000, COL, MRSA252, as well as methicillin-sensitive S.aureus (MSSA) strains Reynolds, Becker, Smith Diffuse, MN8, andvancomycin intermediate sensitive (VISA) strain Mu50, were all obtainedfrom the Network on Antibiotic Resistance in Staphylococcus aureus(NARSA); MSSA strains Newman and Rosenbach were from ATCC.Staphylococcus epidermidis, Bacillus subtilis, Enterococcus faecalis,and Streptococcus pyogenes were obtained from Ward's Natural Science;Listeria monocytogenes was from ATCC. Bacteria were grown on tryptic soyagar (TSA) plates supplemented with 5% sheep blood for 18 h at 37° C.For liquid cultures, single colonies from TSA plates were inoculatedinto tryptic soy broth (TSB) and incubated at 37° C. while shaking at200 rpm for 18 h. Fresh 100-fold dilutions of these cultures in freshTSB were further subcultured for various times.

FACS Analysis of rF1 Binding to Whole Bacteria Grown in vitro.

For antibody staining of whole cells, bacteria were harvested from TSAplates or TSB cultures, and washed with Hank's Buffered Salt Solution(HBSS) without phenol red supplemented with 0.1% BSA (IgG free; Sigma)and 10 mM Hepes, pH 7.4 (HB buffer), by centrifugation at 1700×g for 20min. Bacterial concentrations were estimated by reading optical densityat 630 nm. Bacterial suspensions of 20×10⁸ CFU/mL in HB buffer weremixed with equal volumes of 300 μg/mL of rabbit IgG (Sigma), andincubated for 1 h at room temperature (RT) to block nonspecific IgGbinding. Primary antibodies, including rF1 and a human IgG1 isotypecontrol, were added to a final concentration of 2 μg/mL, and thesemixtures were incubated for 15 mM at RT. After two washes with HB,bacterial pellets were resuspended in a solution of fluorescentanti-human IgG secondary antibodies (Jackson Immunoresearch) andincubated for 15 min at RT. The bacteria were washed twice with PBS,resuspended in PBS with 1% paraformaldehyde, and analyzed by flowcytomery.

FACS Analysis of rF1 Binding to Whole Bacteria from Infected Tissues.

For analysis of antibody binding to bacteria from infected tissue, 4 hsubcultures of USA300 in TSB were washed with PBS. Mice were injectedintravenously with a 100 μL of a suspension of USA300 in PBS with anestimated concentration of 10×10⁸ CFU/mL. Three days later, kidneys,livers, and lungs were harvested and homogenized using conical tissuegrinder tubes (VWR). When indicated, organs were harvested at differenttime points of infection. To lyse mouse cells, the homogenates wereincubated for 10 min at RT in PBS containing 0.1% Triton-X100 (Thermo),10 μg/mL of DNAseI (Roche) and Complete Mini protease inhibitor cocktail(Roche), and passed through a 40 μm filter (Falcon). The cellsuspensions were washed twice with PBS and resuspended in HB buffer,mixed with equal volumes of 600 μg/mL of human IgG (Sigma), andincubated for 1 h at RT. Primary Abs, including rF1 and a human IgG1isotype control, were added to a final concentration of 2 μg/mL. Todifferentiate bacteria from mouse organ debris, rabbit IgG anti-S.aureus (Abcam) was added to a concentration of 20 μg/mL. Afterincubation for 15 min at RT, cells were washed twice with HB buffer, andresuspended in a mixture of anti-human IgG and anti-rabbit IgG secondaryantibodies, each labeled with a different fluorochrome (JacksonImmunoresearch). After two washes with PBS, cells were resuspended inPBS with 2% paraformaldehyde and analyzed by flow cytometry. Afterselecting bacteria from double fluorescence plots by gating for positivestaining with rabbit IgG anti-S. aureus, overlay histogram plots offluorescence intensities of rF1 and isotype control were generated.

Results

rF1 Binds Strongly to 14 S. aureus Strains and S. epidermidis

Seven Methicillin-resistant S. aureus (MRSA) strains, sixmethicillin-sensitive S. aureus (MSSA) strains, one vancomycinintermediate sensitive S. aureus (VISA) strain, S. epidermidis andseveral other gram-positive species were tested for binding to rF1antibody. As shown in FIG. 4, rF1 strongly binds to all 14 S. aureusstrains (FIG. 4A) and to S. epidermidis (FIG. 4B), but not to the othertested gram-positive species (FIG. 4B).

rF1 Binds to MRSA from Different Growth Stages and From in vivoInfection

We tested the ability of mAb rF1 to bind to bacteria both in differenttissues and over the course of infection. We found that rF1 bound tobacteria isolated from murine kidney, liver and lung tissue two daysafter infection and that the binding to bacteria isolated from infectedkidneys was stable, binding bacteria 2, 3 and 8 days after infection.

Binding of rF1 antibody to different growth stages of MRSA (strainUSA300), namely early logarithmical growth (2 hours) and postexponential growth (8 hours) in TSB cultures, and growth of solidcolonies on TSA plates was tested. rF1 strongly binds to all growthstages tested (FIG. 5A).

Binding of rF1 antibody to MRSA (strain USA300) to whole bacteria frominfected tissue was tested in homogenized kidneys from mice three dayafter the mice were systemically infected with MRSA. As shown in FIG. 5BrF1 binds to MRSA obtained from infected tissue.

Example 3

Further experiments were performed to identify the epitope bound byantibody rF1. Although binding to LTA preparations had been observed(see, e.g., Example 1), that binding was not as robust as binding towhole bacteria suggesting that another epitope may be involved in rF1binding.

Methods

Immunoprecipitation, Western Blotting and Mass Spectrometry of Cell WallLysates of S. aureus and S. epidermidis and Commercial WTA Cell WallPreparation

40 micrograms of a commercial wall teichoic acid (WTA) preparation fromWood46 S. aureus strain (Biodesign/Meridian Life Sciences, Maine) wassplit into two parts and immunoprecipitated with 1 ug/ml of rF1 orisotype control human antibody. Antibodies were captured with ProteinA/G Ultralink Resin (Pierce). The samples were then treated with 50 mMdithiothreitol, 10 mM 2-Iodoacetamide and run on an 8% Tris-glycine geland subsequently Western blotted with rF1.

A cell wall preparation of a 20 ml overnight culture of USA300 S. aureusstrain was prepared by treatment of the culture (40 mg cell pellet/ml)with 100 mg/ml of lysostaphin in 30% raffinose buffer at 37° C. for 30minutes. The entire cell wall prep was filtered, diluted up to 10 mlwith NP40 Lysis buffer and incubated twice with anti-Flag M2 Agarose(Sigma) to deplete as much protein-A from the cell wall prep aspossible. Final cell wall preparation was split into two parts andimmunoprecipitated with 1 ug/ml of rF1 or isotype control humanantibody. Antibodies were captured with Protein A/G Ultralink Resin(Pierce). The samples were then treated with 50 mM dithiothreitol, 10 mM2-Iodoacetamide and run on an 8% Tris-glycine gel and subsequentlysilver stained or Western blotted with rF1.

Lysates from a 20 ml overnight culture of Staphylococcus epidermidis wasprepared by bead beating in NP40 Lysis Buffer. The resulting lysatepreparation was diluted to 10 ml with NP40 Lysis Buffer, split into twoparts and immunoprecipitated with 1 ug/ml of rF1 or control antibody.Antibodies were captured with Protein A/G Ultralink Resin (Pierce). Thesamples were then treated with 50 mM dithiothreitol, 10 mM2-Iodoacetamide and run on an 8% Tris-glycine gel and subsequentlysilver stained or Western blotted with rF1.

For proteomic analysis, samples were applied to a precast SDS PAGE minigel and the resolved proteins stained with Coomassie Blue. Gel slicesfrom the region of the gel corresponding to bands visualized by rF1western blot were excised and reduced, alkylated with iodoacetamide, anddigested in situ with trypsin. Resulting tryptic peptides were analyzedby microcapillary reverse-phase liquid chromatography-nano electrospraytandem mass spectrometry on a hybrid linear ion trap Fourier transformion cyclotron resonance mass spectrometer (LTQ-FT; Thermo Fisher) in adata-dependent experiment. Tandem mass spectral results were submittedfor database searching using Mascot software (Matrix Science).

Expression of Exogenous ClfA Expressed in S. aureus and E. coli

S. aureus expression of His-tagged ClfA: The Clumping factor-A (ClfA)gene was PCR amplified from the sequence encoding the signal sequence tothe sequence encoding the glycine in the LPXTG motif from USA300 genomicDNA. A c-terminal His Tag was engineered at the end of the LPXTG motifand ligated into the pTet S. aureus expression vector (pSAS10 fromGenentech). The resulting construct was then electroporated into S.aureus WT RN4220. A 20 ml culture of either the electroporated RN4220 orRN4220 empty (not carrying a pTet expression vector) was inoculated froman overnight culture (starting OD₆₀₀ of 0.15), grown for 1 hr intrypticase soy broth (TSB) and then induced protein expression for 2 hrswith anhydrotetracycline (200 ng/ml). At the end of the inductionperiod, the S. aureus culture was pre-lysed with lysostaphin (50 ug/ml)and then further lysed by bead beating in lysis buffer (150 mM NaCl, 20mM Tris pH 7.5, 1% triton-X, and Roche protease inhibitor EDTA-freetablets). Cleared lysates were then incubated with NiNta resin (Qiagen)in the presence of 10 mM Imidazole for 1 hr at 4° C. to pull-down therecombinant ClfA protein.

E. coli expression of His-tagged Clfa: The Clumping factor-A (ClfA) genewas PCR amplified from the sequence encoding the N-term signal sequence(with the start methionine) to the sequence encoding the glycine in theC-term LPXTG motif from USA300 genomic DNA. The amplified PCR productwas then ligated in frame with a c-terminal His tag into the pET 21b(+)E. coli expression vector (Novagen). The resulting construct wastransformed into E. coli BL21-gold (DE3) Competent Cells (Stratagene)and induced for protein expression for 3.5 hrs with IPTG according tothe manufacturer's instructions. The induced E. coli culture was lysedby bead beating in Lysis Buffer (150 mM NaCl, 20 mM Tris pH 7.5, 1%triton-X, and Roche protease inhibitor EDTA-free tablets). Clearedlysates were then incubated with NiNta resin (Qiagen) in the presence of10 mM Imidazole for 1 hr at 4° C. to pull-down the recombinant ClfAprotein.

Expression of Exogenous ClfA Expressed in E. coli and Incubation with S.aureus Lysate_(—)

Expression and Purification of S. aureus cell surface SDR proteins ClfA,ClfB, SdrC, SdrD, SdrE in E. coli: The ClfA, ClfB, SdrC, SdrD and SdrEgenes were PCR amplified from the sequence encoding the mature start ofthe protein to the sequence encoding the glycine in the LPXTG motif fromUSA300 genomic DNA and ligated in-frame with an N-terminal Unizyme taginto the ST239 Vector (Genentech). The constructs were transformed intoE. coli 58F3 (Genentech), induced for protein expression andsubsequently purified.

NiNta capture of NT Unizyme tagged SDR proteins: 500 ug of purifiedN-terminal Unizyme tagged SDR proteins (ClfA, ClfB, SdrC, SdrD, SdrE)were diluted in PBS containing protease inhibitors (EDTA-free) andincubated with NiNta resin (Qiagen) for 1.5 hr at 4° C. NiNta resin withthe captured Unizyme tagged SDR protein was then washed once with washbuffer (50 mM NaH₂PO₄, 300 mM NaCl; pH 8.0).

Modification of E. coli produced SDR proteins by S. aureus lysate: A 25ml culture was started from an overnight culture of ΔPan Sdr mutant(ClfA-ClfB-SdrCDE-null; gift of Tim Foster, Trinity College Dublin)Newman S. aureus (starting OD₆₀₀ of 0.15) and grown in TSB for 3 hrs(exponential phase) 37° C., 200 rpm. The exponential phase culture wasthen resuspended in 1 ml of PBS and lysed with 200 ug/ml lysostaphin inthe presence of 250 units of Benzonase Nuclease (Novagen) at 37° C. for30 minutes. The lysates were cleared of debris by spinning down atmaximum speed in a micro-centrifuge for 10 minutes at 4° C. Modificationof NiNta captured E. coli SDR protein was carried out by incubation withcleared ΔPan Sdr mutant S. aureus lysates for 1 hr at 37° C.

Western Blotting of modified E. coli SDR protein: Non-modified ormodified NiNta captured E. coli SDR protein samples were then washed 3×with wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM Imidazole; pH 8.0),prepared for Western Blotting and run on an 8% Tris Glycine Gel(Invitrogen). Western blots were blotted with either the rF1 antibody oran anti-Unizyme antibody (Genentech).

Identification of SDR Domain as Antigen for rF1

S. aureus expression of MBP-SD Constructs: The maltose binding protein(MBP) gene was PCR amplified from the pMAL-c5x vector (New EnglandBioLabs [NEB], Ipswich, Mass., USA) from the sequence encoding the startof the mature protein until the sequence encoding the end of the FactorXa cleavage site. The varying lengths of c-terminal His tagged SD (SD,SDS, DSD, SDSD, SDSDS, SDSDSD) were synthesized as single strandedoligonucleotides and annealed together to make double stranded dna. TheSdr region of the ClfA gene was PCR amplified from the sequence encodingthe beginning (560D) of the Sdr region and included up to either thesequence encoding 618A or 709S of the SD region followed by the dnasequence coding for a His Tag from the respective plasmidspTet.ClfA.SD618A or pTet.ClfA.SD709S (Genentech). As a control, thesequence encoding the A domain of the ClfA gene from the beginning ofthe mature protein until the sequence encoding the end of the A domain(538G) followed by the sequence coding for a His Tag was PCR amplifiedfrom the plasmid pTet.ClfA.Adom.538G (Genentech). The MBP insert alongwith one of the various SD inserts or ClfA A domain were ligated intothe pTet S. aureus expression vector (pSAS10 from Genentech). Theresulting constructs were then electroporated into the S. aureus RN4220Δsortase (sortase deletion mutant strain in the RN4220 background).

A 20 ml culture of either the electroporated RN4220Δ sortase or RN4220Δsortase empty (not carrying a pTet expression vector) was inoculatedfrom an overnight culture (starting OD₆₀₀ of 0.15), grown for 1 hr intrypticase soy broth (TSB) supplemented with glucose (2 g/L) and theninduced for protein expression for 2 hrs with anhydrotetracycline (200ng/ml). At the end of the induction period, the S. aureus culture wasresuspended in Column Buffer (150 mM NaCl, 20 mM Tris pH 7.5 and Rocheprotease inhibitor EDTA-free tablets) and lysed with 200 ug/mllysostaphin in the presence of 250 units of Benzonase Nuclease (Novagen)at 37° C. for 30 minutes. The lysates were cleared of debris by spinningdown at maximum speed in a micro-centrifuge for 10 minutes at 4° C. Thecleared lysates were then incubated with amylose resin (NEB) along witha final EDTA concentration of 1 mM for 1.5 hrs at 4° C. to capture theexpressed MBP-SD proteins.

Western Blotting of S. aureus expressed MBP-SD Constructs: Amylose resinwith the captured MBP-SD proteins were washed three times with ColumnBuffer, further prepared for Western blotting analysis and run on an 8%Tris-glycine gel. Western blots were blotted with either the rF1antibody, an anti-penta His antibody (Qiagen) or an anti-MBP antibody(NEB).

Results

rF1 Reacts to a Unique Family of SDR (Ser-Asp-repeat) Proteins in S.aureus and S. epidermidis

A commercial teichoic acid preparation and cell wall lysate (WTA) of S.aureus (USA300 strain) was tested for binding to rF1 afterimmunoprecipitation. rF1 binds to several components of the commercialteichoic acid preparation (FIG. 6A, left panel) and of the cell walllysate of S. aureus (FIG. 6B, left panel). Using mass spectrometry thesecomponents of the WTA preparation and cell wall lysate were identifiedas ClfA (SdrA), ClfB (SdrB), SdrC, SdrD and SdrE (FIG. 6A, right paneland FIG. 6B, right panel)

A cell wall lysate of S. epidermidis was tested for binding to rF1 afterimmunoprecipitation. FIG. 6C (left panel) shows binding of rF1 toseveral components of a cell wall lysate of S. epidermidis. Thesecomponents were not identified with a control antibody. Using massspectrometry these cell wall components were identified as SdrF, SdrGand SdrH (FIG. 6C right panel).

Exogenous ClfA expression in S. aureus and E. coli

Exogenous ClfA expressed in S. aureus is reactive to rF1, whereas ClfAexpressed in E. coli was not (FIG. 7A). However, incubation of E. coliexpressed Sdr proteins ClfA (SdrA), ClfB (SdrB), SdrC, SdrD and SdrEwith S. aureus lysate regained rF1 reactivity (FIG. 7B).

rF1 Binds to SDR Domains Expressed in S. aureus

rF1 antibody binds to ClfA Sdr regions consisting of the ClfA 560D-618Sand ClfA 560D-709S (FIG. 8) expressed in S. aureus. rF1 does not bind tothe A-domain of ClfA or to small peptide sequences consisting of up tothree SD repeats.

Example 4 Methods Cloning of the Variable Regions of rF1 Into pRK Vector

Phusion DNA polymerase, restriction enzymes EcoRV, KpnI, PvuII, ApaI,AgeI, and AhdI, T4 DNA ligase were purchased from New England BioLabs,Ipswich, Mass., USA. Pfu DNA polymerase, Quick change II Site-DirectedMutagenesis Kit were purchased from Stratagene/Agilent Technologies,Santa Clara, Calif., USA. 2% agarose gel was purchased from Invitrogen,Carlsbad, Calif., USA. pRK vectors pRK.LPG3.HuKappa and pRK.LPG4.HumanHCwere obtained from Genentech/Roche, South San Francisco, Calif., USA.

The variable domains of heavy and light chains of rF1 Mab from the pCPEOvectors were placed into the pRK mammalian expression vectors.Polymerase chain reaction (PCR) was performed in a total volume of 50 μlusing Pfu DNA polymerase according to standard PCR procedures. V_(H) wasamplified with the primers YiHCF) 5′-ATG GCT GAG GTG CAG CTG GTG GAG TCTG-3′ (SEQ ID NO:18) and YiHCR2 5′-GAA CAC GCT GGG GCC CTT GGT GCT GGCACT CGA GAC TGT GAC CAG GGT GCC AGG T*CC CCA G-3′ (SEQ ID NO:19) (therestriction sites of PvuII and ApaI are underlined and the * indicatesthe single nucleotide G to T change to eliminate the internal ApaI site)and V_(L) was amplified with the primers YiLCF 5′-CGG CTC GAC CGA TATCCA GCT GAC CCA GAG-3′ (SEQ ID NO:20) and YiLCR 5′-GAT TTC CAG CTTGGTACC CTG GCC G-3′ (SEQ ID NO:21) (the EcoRV and KpnI are underlined).Unique PCR products with 393 (V_(H)) and 328 (V_(L)) bp, respectively,were directly digested by PvuII and ApaI for V_(H), EcoRV and KpnI forV_(L), followed by Gel purification (Invitrogen, Catalog# K2100-12).V_(H) and V_(L) were individually ligated into pRK vector fragments ofpRK/PvuII, ApaI for Heavy chain and pRK/EcoRV, KpnI for light chainusing T4 DNA ligase. The DNA plasmids were confirmed by restrictionenzyme digestion patterns, i.e. rF1 V_(H) released ˜300 by and 5.8 kbbands by AgeI, whereas rF1V_(L) shown 2.3 and 3.1 kb bands by AhdI on 2%agarose gel.

Chemical Stress Testing

By comparing antibody primary sequence data to experimentally observeddegradation events, it has become clear that certain sequence motifs maybe prone to degradation (i.e, aspartate isomerization at DD, DG, and DSsequences and asparagine deamidation at NG sequences). If such adegradation “hot spot” appears in the CDR of an antibody, binding andpotency may be negatively impacted. For molecules containing hot spots,chemical stress testing provides a way to assess the susceptibility ofthese motifs to degradation by putting the antibody into a platformformulation buffer and comparing a control sample to one stressed at 40°C. for two weeks. If degradation is observed the primary sequence can bere-engineered to remove the hot spot.

After the sample is stressed for two weeks at 40° C., a variety ofanalytical assays are performed to assess where and how much degradationoccurred. An imaged capillary isoelectric focusing (icIEF) analysis wasperformed to examine charge variants and the intact and reduced antibodymass was verified using mass spectrometry, and an LC-MS/MS peptide mapwas performed to obtain site-specific degradation information.

Mutagenesis

The rF1 pRK plasmids underwent a series of site-directed mutagenesis tostabilize the rF1 Mab by N (AAC) 53S (AGC) in heavy chain CDR2 withusing FHV 5′-GGT GGC CAG CAT CAA CAG CGG CAA CAA CCC CTA CTA CG-3′ (SEQID NO:22) and RHV 5′-CGT AGT AGG GGT TGT TGC CGC TGT TGA TGC TGG CCACC-3′ (SEQ ID NO:23) (mutagenesis site is underlined) via Quik change IISite-Directed Kit. The mutagenesis variant was sequenced.

Results

The primary sequence of antibody rF1 contained one potential asparaginedeamidation site in light chain CDR2: NNGNN (SEQ ID NO:24). Stresstesting revealed an increase of deamidation in stressed samples at N53(numbering according to Kabat, 1991). Site-directed mutagenesis wasperformed to stabilize the rF1 Mab by N (AAC, (SEQ ID NO:25)) to 53S(AGC, (SEQ ID NO:26)) substitution in heavy chain CDR2. Sequencing ofthe mutagenesis variant confirmed an N53S mutation.

Example 5 Development of Cys Variants for Antibody-Drug Conjugate (ADC)Applications

The rF1 pRK plasmids underwent a series of site-directed mutagenesiswith oligos of rF1pRK.LC(205/210)VCF (468075) 5′-GGG CCT GAG CTC GCC CTGCAC AAA GAG CTT CAA CAG-3′ (SEQ ID NO:27) and rF1pRK.LC(205/210)VCR(468076) 5′-CTG TTG AAG CTC TTT GTG CAG GGC GAG CTC AGG CCC-3′ (SEQ IDNO:28) (Cys mutant is underlined) to generate the light chain linkers ofrF1 thioMab, and rF1pRK.HCN53S.A121CF (468464) 5′-CTG GTC ACA GTC TCGAGT TGC AGC ACC AAG GGC CCA TC-3′ (SEQ ID NO:29) andrF1pRK.HCN53S.A121CR (468465) 5′-GAT GGG CCC TTG GTG CTG CAA CTC GAG ACTGTG ACC AG-3′ (SEQ ID NO:30) (Cys mutant is underlined) to make theheavy chain linkers of rF1 thioMab via the Stratagene's Site-Directedmethod. Light chain V205C and heavy chain A114C were confirmed bysequencing of the variants (FIG. 9).

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We claim:
 1. An isolated antibody comprising: (a) a heavy chain CDR1sequence RFAMS (SEQ ID NO:1), and (b) a heavy chain CDR2 sequenceSINNGNNPYYARSVQY (SEQ ID NO:2), and (c) a heavy chain CDR3 sequenceDHPSSGWPTFDS (SEQ ID NO:3).
 2. The antibody of claim 1 furthercomprising a variable light chain.
 3. An isolated antibody comprising:(a) a light chain CDR1 sequence RASENVGDWLA (SEQ ID NO:4), and (b) alight chain CDR2 sequence KTSILES (SEQ ID NO:5), and (c) a light chainCDR3 sequence having at least 88% identity to QHYXRFPYT, wherein X isany amino acid.
 4. The antibody of claim 3 wherein X is a hydrophobicamino acid selected from the group consisting of I, M, A, V, L, F, Y,and W.
 5. The antibody of claim 4 wherein the light chain CDR3 sequencehas a sequence of QHYXRFPYT (SEQ ID NO:6), wherein X is I or M.
 6. Theantibody of claim 3 further comprising a variable heavy chain.
 7. Theantibody of claim 1 further comprising: (d) a light chain CDR1 sequenceRASENVGDWLA (SEQ ID NO:4), and (e) a light chain CDR2 sequence KTSILES(SEQ ID NO:5), and (f) a light chain CDR3 sequence having at least 88%identity to QHYXRFPYT, wherein X is any amino acid.
 8. The antibody ofclaim 7 wherein X is a hydrophobic amino acid selected from the groupconsisting of I, M, A, V, L, F, Y, and W.
 9. The antibody of claim 8wherein the light chain CDR3 sequence has a sequence of QHYXRFPYT (SEQID NO:6), wherein X is I or M.
 10. The antibody of claim 3 wherein thelight chain CDR3 sequence is QHYIRFPYT (SEQ ID NO:6).
 11. The antibodyof claim 7 wherein the light chain CDR3 sequence is QHYMRFPYT (SEQ IDNO:6).
 12. The antibody of claim 1 wherein the antibody comprises avariable heavy chain sequence comprising: (a) framework 1 (FW1) whichhas at least 90% identity to EVQLLESGGGLVQPGGSLRLSCAASGFTLS (SEQ ID NO:37); (b) framework 2 (FW2) which has at least 90% identity toWVRQAPGRGLEWVA (SEQ ID NO: 39); (c) framework 3 (FW3) which has at least90% identity to RFTVSRDVSQNTVSLQMNNLRAEDSATYFCAK (SEQ ID NO: 41); and(d) framework 4 (FW4) which has at least 90% identity to WGPGTLVTVSS(SEQ ID NO: 43).
 13. The antibody of claim 12 wherein the antibody has avariable heavy chain sequence comprising: (a) framework 1 (FW1) whichhas the sequence of EVQLLESGGGLVQPGGSLRLSCAASGFTLS (SEQ ID NO: 37); (b)framework 2 (FW2) which has the sequence of WVRQAPGRGLEWVA (SEQ ID NO:39); (c) framework 3 (FW3) which has the sequence ofRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAK (SEQ ID NO: 41); and (d) framework 4(FW4) which has the sequence of WGPGTLVTVSS (SEQ ID NO: 43).
 14. Theantibody of claim 1 wherein the antibody comprises a variable heavychain sequence comprising a sequence which has at least 90% sequenceidentity to the sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ IDNO:7).
 15. The antibody of claim 14 wherein the antibody comprises avariable heavy chain having a sequence ofEVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ IDNO:7).
 16. The antibody of claim 3 wherein the antibody has a variablelight chain sequence comprising: (a) framework 1 (FW1) which has atleast 90% identity to DIQLTQSPSALPASVGDRVSITC (SEQ ID NO: 47); (b)framework 2 (FW2) which has at least 90% identity to WYRQKPGKAPNLLIY(SEQ ID NO: 49); (c) framework 3 (FW3) which has at least 90% identityto GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC (SEQ ID NO: 51); and (d) framework 4(FW4) which has at least 90% identity to FGQGTKLEIKRTV (SEQ ID NO: 53).17. The antibody of claim 16 wherein the antibody has a variable lightchain sequence comprising: (a) framework 1 (FW1) which has the sequenceof DIQLTQSPSALPASVGDRVSITC (SEQ ID NO: 47); (b) framework 2 (FW2) whichhas the sequence of WYRQKPGKAPNLLIY (SEQ ID NO: 49); (c) framework 3(FW3) which has the sequence of GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC (SEQ IDNO: 51); and (d) framework 4 (FW4) which has the sequence ofFGQGTKLEIKRTV (SEQ ID NO: 53).
 18. The antibody of claim 16 wherein theantibody has a variable light chain sequence comprising: (a) framework 1(FW1) which has the sequence of DIQLTQSPSALPASVGDRVSITC (SEQ ID NO: 47);(b) framework 2 (FW2) which has the sequence of WYRQKPGKAPNLLIY (SEQ IDNO: 49); (c) framework 3 (FW3) which has the sequence ofGVPSRFSGSGSGTEFTLTISSLQPDDFATYYC (SEQ ID NO: 51); and (d) framework 4(FW4) which has the sequence of FGQGTKVEIKRTV (SEQ ID NO: 59).
 19. Theantibody of claim 12 further comprising (a) a light chain CDR1 sequenceRASENVGDWLA (SEQ ID NO:4), (b) a light chain CDR2 sequence KTSILES (SEQID NO:5), and (c) a light chain CDR3 sequence QHYXRFPYT (SEQ ID NO:6),wherein X is I or M; (d) framework 1 (FW1) which has at least 90%identity to DIQLTQSPSALPASVGDRVSITC (SEQ ID NO: 47); (e) framework 2(FW2) which has at least 90% identity to WYRQKPGKAPNLLIY (SEQ ID NO:49); (f) framework 3 (FW3) which has at least 90% identity toGVPSRFSGSGSGTEFTLTISSLQPDDFATYYC (SEQ ID NO: 51); and (g) framework 4(FW4) which has at least 90% identity to FGQGTKLEIKRTV (SEQ ID NO: 53).20. The antibody of claim 19 wherein the antibody has a variable heavychain sequence comprising: (a) framework 1 (FW1) which has the sequenceof EVQLLESGGGLVQPGGSLRLSCAASGFTLS (SEQ ID NO: 37); (b) framework 2 (FW2)which has the sequence of WVRQAPGRGLEWVA (SEQ ID NO: 39); (c) framework3 (FW3) which has the sequence of RFTVSRDVSQNTVSLQMNNLRAEDSATYFCAK (SEQID NO: 41); and (d) framework 4 (FW4) which has the sequence ofWGPGTLVTVSS (SEQ ID NO: 43) and wherein the antibody has a variablelight chain sequence comprising: (a) framework 1 (FW1) which has thesequence of DIQLTQSPSALPASVGDRVSITC (SEQ ID NO: 47); (b) framework 2(FW2) which has the sequence of WYRQKPGKAPNLLIY (SEQ ID NO: 49); (c)framework 3 (FW3) which has the sequence ofGVPSRFSGSGSGTEFTLTISSLQPDDFATYYC (SEQ ID NO: 51); and (d) framework 4(FW4) which has the sequence of framework 4 (FW4) which has the sequenceof FGQGTKLEIKRTV (SEQ ID NO: 53) or FGQGTKVEIKRTV (SEQ ID NO: 59). 21.The antibody of claim 3 wherein the antibody comprises a variable lightchain sequence which has at least 90% sequence identity to the sequenceDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKVEIKRTV, wherein X is I or M(SEQ ID NO:11).
 22. The antibody of claim 21 wherein the antibodycomprises a variable light chain having a sequence ofDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKVEIKRTV, wherein X is I or M(SEQ ID NO:11).
 23. The antibody of claim 7 wherein antibody comprises:(a) a variable heavy chain comprising a sequence ofEVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFD SWGPGTLVTVSS (SEQID NO:7) and (b) a variable light chain comprising a sequence ofDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKVEIKRTV, wherein X is Ior M (SEQ ID NO:11).
 24. The antibody of claim 7 wherein antibodycomprises: (a) a variable heavy chain comprising a sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFD SWGPGTLVTVSS (SEQID NO:9) (b) a variable light chain comprising a sequence ofDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRTV, wherein X is Ior M (SEQ ID NO:8).
 25. The antibody of claim 7 wherein antibodycomprises: (a) a variable heavy chain comprising a sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFD SWGPGTLVTVSS (SEQID NO:9) (b) a variable light chain comprising a sequence ofDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRA, wherein X is I orM (SEQ ID NO:10).
 26. The antibody of claim 7 wherein antibodycomprises: (a) a variable heavy chain comprising a sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFD SWGPGTLVTVSS (SEQID NO:9) and (b) a variable light chain comprising a sequence ofDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKVEIKRTV, wherein X is Ior M (SEQ ID NO:11).
 27. The antibody of claim 7 wherein antibodycomprises: (a) a variable heavy chain comprising a sequence ofEVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFD SWGPGTLVTVSS (SEQID NO:7) and (b) a variable light chain comprising a sequence ofDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRTV, wherein X is Ior M (SEQ ID NO:8).
 28. The antibody of claim 7 wherein antibodycomprises: (a) a variable heavy chain comprising a sequence ofEVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFD SWGPGTLVTVSS (SEQID NO:7) and (b) a variable light chain comprising a sequence ofDIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRA, wherein X is I orM (SEQ ID NO:10).
 29. The antibody of claim 7 wherein antibodycomprises: (a) a heavy chain comprising a sequence of SEQ ID NO: 55 and(b) a light chain comprising a sequence of SEQ ID NO:
 57. 30. Theantibody of claim 7 wherein antibody comprises: (a) a heavy chaincomprising a sequence of SEQ ID NO: 56 and (b) a light chain comprisinga sequence of SEQ ID NO:
 58. 31. The antibody according to claim 7,wherein an asparagine at position 53 in the variable heavy chain hasbeen replaced by another amino acid, whereby the amino acid numbering isaccording to Kabat (1991).
 32. The antibody according to claim 29,wherein said asparagine at position 53 is replaced with a serine,wherein an alanine at position 114 is replaced with a cysteine, andwherein an arginine at position 222 is replaced with a lysine, wherebythe amino acid numbering is according to Kabat (1991).
 33. The antibodyaccording to claim 29 wherein valine at variable light chain position205 is replaced with cysteine, an alanine at variable light chainposition 110 is replaced with valine, and a leucine at variable lightchain position 104 is replaced with a valine, whereby the amino acidnumbering is according to Kabat (1991).
 34. The antibody according toclaim 1 wherein one or more CDR is optimized to improve binding efficacyor stability.
 35. The antibody according to claim 3 wherein one or moreCDR is optimized to improve binding efficacy or stability.
 36. Theantibody according to claim 7 wherein one or more CDR is optimized toimprove binding efficacy or stability.
 37. The antibody according toclaim 1 further comprising at least one framework region wherein atleast one sequence in at least one framework region is optimized toimprove binding efficacy or stability.
 38. The antibody according toclaim 3 further comprising at least one framework region wherein atleast one sequence in at least one framework region is optimized toimprove binding efficacy or stability.
 39. The antibody according toclaim 7 further comprising at least one framework region wherein atleast one sequence in at least one framework region is optimized toimprove binding efficacy or stability.
 40. The antibody according toclaim 1 further comprising at least one framework region wherein atleast one sequence in at least one framework region is optimized tominimize immunogenicity.
 41. The antibody according to claim 3 furthercomprising at least one framework region wherein at least one sequencein at least one framework region is optimized to minimizeimmunogenicity.
 42. The antibody according to claim 3 further comprisingat least one framework region wherein at least one sequence in at leastone framework region is optimized to minimize immunogenicity.
 43. Anisolated or recombinant antibody producing cell capable of producing theantibody or functional part or immunoglobulin chain or functionalequivalent according to claim 7.